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FHYSIOLO'GICAL SCIENCES

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INTERNATIONAL SERIES OF MONOGRAPHS ON
PURE AND APPLIED BIOLOGY

Division: MODERN TRENDS IN PHYSIOLOGICAL SCIENCES

General Editors : P. Alexander and Z. M, Bacq

Volume 1

UNITY AND DIVERSITY
IN BIOCHEMISTRY

OTHER TITLES IN THE SERIES ON PURE AND APPLIED BIOLOGY

Modern Trends in Physiological Sciences Division
Vol. 2. BRACKET — The Biochemistry of Development
Vol. 3. GEREBTZOFF—Cholinesterases
Vol. 4. BROVHA— Physiology in Industry

Biochemistry Division

Vol. 1. PITT-RIVERS and TATA— The Thyroid Hormones

Botany Division

Vol. 1. BOR — Grasses of Burma, Ceylon, India and Pakistan

Vol. 2. TURRILL (Ed.)— Vistas in Botany

Vol. 3. SHULTES— Orchids of Trinidad and Tobago

Zoology Division

Vol. 1 . RAVEN — An Outline of Developmental Physiology

Vol. 2. RAVEN — Morphogenesis: The Analysis of Molluscan Development

Vol. 3. SAVORY — Instinctive Living

Vol. 4. KERKUT— Implications of Evolution

Vol. 5. JENKIN— .<4mma/ Hormones

UNITY AND DIVERSITY
IN BIOCHEMISTRY

An Introduction to Chemical Biology

hy
MARCEL FLORKIN

University of Liege
Translated from the French by

T.WOOD

University of Sydney

PERGAMON PRESS

NEW YORK • OXFORD • LONDON • PARIS

1960

PERGAMON PRESS INC.

122 East 55th Street, New York 22, N. Y.
P.O. Box 47715, Los Angeles, California

PERGAMON PRESS LTD.

Headington Hill Hall, Oxford
4 & 5 Fitzroy Square, London W.l.

PERGAMON PRESS S.A.R.L.
24 Rue des Ecoles, Paris F«

PERGAMON PRESS G.m.b.H.

Kaiserstrasse 75, Frankfurt am Main

Copyright

®
1960

Marcel Florkin

First Published in English 1960

Library of Congress Card Number 58-12669

PRINTED IN GREAT BRITAIN BY THE BAY TREE PRESS, HERTS.

•SS5

Contents

PAGE

Translator's Preface ix

Introduction xi

PART ONE

Chapter I: The Biosphere 1

Chapter II: Constituents of the Biosphere 7

I. The three principal biochemical structures 8

II. Other chemical structures of general interest 30

Chapter III: Modes of Linkage by Covalent Bonds 57

I. "Oside" Linkage . 57

II. Ester linkage (and anhydride linkage) 61

III. Peptide bond 77

Chapter IV: Macromolecules 83

I. Polysaccharides 85

II. Proteins 93

III. Nucleoproteins 107

IV. Metalloproteins 111
V. Lipoproteins 127

PART TWO— ENZYMES AND BIOCHEMICAL ENERGETICS

Chapter I: General Principles of Biochemistry 131

I. Free energy 131

II. Energy coupling 136

III. Energy rich bonds 137

IV. The phosphate cycle 138
V. Biological oxido-reductions and the generation of energ>'-rich bonds 138

VI. The cellular dynamo 145

VII. The pyrophosphate bond and cellular work 146

VIII. Chemical equilibrium and the stationary state 147

Chapter II: Enzymes 151

I. Definition 151

II. Enzymes and activators 152

III. Classification of enzymes 152

IV. Kinetics 166
V. The mechanism of action of enzymes and coenzymes 171

PART THREE— CHEMICAL REACTIONS IN THE BIOSPHERE

Introduction 179

Chapter I: Destructive and Non-destructive Methods in Modern Biochemistry 181

I. From the whole organism to the pure enzyme 181

II. Biochemical investigation and the use of isotopes 182

III. The use of mutant strains of micro-organisms 184

vi CONTENTS

Chapter II: Priming Reactions 186

I. Glycolysis and the hexosemonophosphate shunt 186

II. Another pathway for the production of acetyl-CoA: the fatty acid cycle 196

III. The tricarboxylic acid cycle 199

IV. Respiratory chains 207
V. Mechanisms for the breakdown of amino acids 209

VI. Interrelations between priming reactions 223

VII. Energetics of the priming reactions 223

Chapter III: Biosyntheses 229

I. The materials for biosyntheses 229

II. Biosynthesis 230

PART FOUR— TOPOBIOCHEMISTRY AND CELLULAR REGULATION

Chapter I: Cellular Topochemistry 273

Chapter II: Cellular Regulation 283

I. Factors which determine the velocity and the path of enzymatic reaction chains 283

II. The Pasteur effect 285

III. The genetic control of the relative rates of enzymatic reactions 286

PART FIVE— BIOCHEMICAL DIVERSITY

Chapter I: Some Aspects of Biochemical Diversity 289

I. Terpenes 289

II. Porphyrins 291

III. Protein macromolecules 301

Chapter II: The Inheritance of Biochemical Characteristics 304

I. Control of biochemical characteristics by genes 304

II. Biochemical differentiation of cells in a single organism 305

III. Phenotype and "milieu" 315

Chapter III: Biochemisty and Taxonomy 317

I. Biochemical diversity 317

II. Diversity within species 323

III. Diversity between species 323

Chapter IV: Biochemical Evolution 333

I. Definition 333

II. Evolution of biochemical constituents 333

III. Evolution of biochemical systems 333

PART SIX— THE METABOLISM OF THE BIOSPHERE 339

Introduction 349

Chapter I: Entry into the Biosphere 351

I. Carbon and energy 351

II. Phosphorus 361

III. Nitrogen 362

IV. Sulphur 364

Chapter II: Departure from the Biosphere 366

I. Ammonification in the soil 366

II. The exit of carbon from the biosphere 368

Chapter III: The Cycles 371

I. The carbon cycle 371

II. The nitrogen cycle 371

III. The sulphur cycle 376

IV. The circulation of phosphorus 377
V. The metabolism of the biosphere 377

Index 381

LIST OF ABBREVIATIONS

ADP

AMP

ATP

DNA

DPN, DPNH

DPT

FAD

FMN

FP

F-6-P

F-i,6-PP

Gal-i-P

G-i-P

G-i,6-PP

G-6-P

HDP

HMP

LTPP

PGA

PGAD

PRPP

Ru-P

Ru-PP

TPN, TPNH

UDP

UDPG

UMP

UTP

Adenosine diphosphate

Adenosine monophosphate

Adenosine triphosphate

Deoxyribonucleic acid

Diphosphopyridine nucleotide

Diphosphothiamine

Flavin adenine dinucleotide

Flavin mononucleotide

Flavin phosphate

Fructose-6-phosphate

Fructose- 1, 6-diphosphate

Galactose- 1 -phosphate

Glucose- 1 -phosphate

Glucose- 1, 6-diphosphate

Glucose-6-phosphate

Heptulose diphosphate

Heptulose monophosphate

Lipothiamide-pyrophosphate

3-Phosphoglyceric acid

Phosphoglyceraldehyde dehydrogenase

Phosphoribosylpyrophosphate

Ribulose-5-phosphate

Ribulose diphosphate

Triphosphopyridine nucleotide

Uridine diphosphate

Uridine diphosphate glucose

Uridine monophosphate

Uridine triphosphate

TR.\NSLATOR'S PREFACE

The translation of Professor Florkin's book has been complicated by the
rapid progress of biochemistry in the last few years, necessitating many
alterations and additions to the original text, to keep it up to date and to
include new material. The subject-matter dealt with in these pages
covers a very wide field, and I would crave the indulgence of the reader
for any errors due to lack of familiarity with some of the topics discussed.
Part of this discussion is at a philosophical level and, although not having
had the pleasure of meeting Professor Florkin personally, I have tried to
convey as faithfully as possible the ideas and concepts set out in the original
text. When in doubt, I have stayed as closely as possible to the phrase-
ology of the original. I have retained the term "biosphere", for example,
to describe the total collection of living organisms. Enzyme nomenclature,
too, has been a problem, but when in doubt I have used the terms given
in Baldwin's Dynamic Aspects of Biochemistry , or alternatively, the simplest
and most descriptive name for the enzyme. I have endeavoured to write
in good concise English and I trust that the result is clear and readable.

Terry Wood

IX

INTRODUCTION

In these pages, the reader will find neither a treatise nor a textbook on
biochemistry, but a number of essays grouped around ideas of the unity and
diversity of organisms in the biochemical sphere, "The manifold and the
one" are eternal preoccupations of the human intellect, and we must not be
surprised that, from the time biochemistry has been able to gather together
a sufficient number of facts, the search for the lowest common denominator
of all organisms or a "unity of biochemical plan" has been confused in
many minds with the idea of a comparative biochemistry. The latter is a
problem which is perhaps more relevant to natural philosphy than to
scientific investigation, for we are becoming more and more aware of the
extreme diversity of biochemical function arising during cellular differentia-
tion in a single organism, as well as in the multiplicity of species and even
of individuals. The biosphere, by which we understand the total amount
of living matter, behaves like a chemist of a very special type. All the
organic compounds present in the many regions of the biosphere and re-
sulting from its biosynthetic activities have structures lying within
certain definite limits. The first part of this book provides a concise
catalogue of these structures but is not coincident with the contents of a
textbook of organic chemistry provided that the latter is not defined as it
was by Berzelius at the beginning of the 19th century, when he wrote that
organic chemistry is that section of physiology describing the composition
of living things and the chemical reactions going on therein. This defini-
tion of organic chemistry is no longer valid today; beginning with the
synthesis of a naturally occurring substance, urea, organic chemistry has
extended its domain to the synthesis of a tremendous number of non-
natural substances. One of the objectives of biochemistry is to define
and understand the nature of the collection of compounds composing
living matter and to distinguish them from those originating from non-
living sources and human inventiveness, all of which are described by the
broad generalizations of chemistry.

The biosphere is not only a chemist of a special type, but also one of
great antiquity whose methods have been developed over a long period of
time since long before there were laboratories of organic chemistry, and
are of an efficiency far from being paralleled in these laboratories. This
point is developed further in the two essays which make up Parts 2 and 3
of this book with the intention of demonstrating the originality of this
organic chemist who has laboured since the dawn of time and comparing
his methods with those of the laboratory chemist. The essays making up

XU UNITY AND DIVERSITY IN BIOCHEMISTRY

Parts 1, 2 and 3 are devoted to general aspects of the biosphere, i.e. to
the biochemical facts common to living beings and which constitute
their lowest common denominator, or, their "unity of biochemical plan".
The cellular theory, as proposed by Theodor Schwann in 1836, taught
that organisms are formed not only of cells, of modified cells and cellular
products, but that a multicellular organism has two levels of individuality,
one on the cellular level and one at the level of the whole organism. In
its final perfected form, the cellular theory recognizes that each cell is
derived from a pre-exisiting cell. The results of biochemical research
have taught us that the manifestations of an underlying biochemical unity
are present in each cell, according to a topochemistry briefly described in
Part 4. In this context, the "unity of plan" is simply the cellular theory
from a biochemical point of view; in the same way as they are units of
structure, cells are units of metabolism. The unity of a structure and
cellular metabolism is only another expression of cellular continuity and the
persistence in this continuity of a definite collection of genes controlling the
synthesis of the collection of enzymes present in each cell. However, no cell
is limited by the underlying biochemical unity, for this is only the canvas
on to which the cell can embroider the numerous variations constituting
its own biological nature, the "unity of plan" remaining an abstraction.

In Part 5 it is only possible to provide a few very brief examples of bio-
chemical variation and biochemical evolution. The few cases quoted
will enable the reader to locate some aspects of biochemical variation at
the level of cellular diflferentiation and at the taxonomic level in the same
organism. These examples show that the biochemical manifestations of
variation are founded on the extension of the general processes of cellular
biochemistry and constitute atypical expressions of general metabolic
systems, variations on each theme being more or less pronounced but
fitting in with general ideas of variation of genotype and biochemical
adaptation of the organism to its surroundings.

Part 6 presents the idea of the metabolism of the biosphere as a whole
and illustrates how this metabolism, like that of each organism, consists
of an entry and an exit of energy and material, but now situated at the
frontier between the biosphere and the inorganic world. The metabolism
of the biosphere is conditioned by manifestations of biochemical variation,
without which life would disappear. In fact, the unity of the biochemical
plan of organisms comes down finally to a continuity of the biosphere
in time and space and the accompanying biochemical diversity which has
appeared as biochemical evolution has progressed with the extension of
the biosphere. This extension has only been possible when, by means of
new ecosystems, the exchanges between the inorganic surroundings and
the biosphere, which are a condition for the survival of the latter, have
been maintained.

INTRODUCTION Xlll

Biochemistry has provided explanations in many fields of physiology
and we can now perceive the preliminary signs of similar progress in a
field that up to now has been outside the scope of biochemical explanation —
that of electrophysiology. The demystification of biology will be a long
and arduous task which is only just beginning. Much work remains to be
done before the natural order, natural selection, adaptation, evolutionary
tendencies, orthogenesis, morphogenesis, etc., are replaced by a know-
ledge of the reality underlying these somewhat poetic terms.

The author is conscious of the imperfections of his book and criticisms
which could be levelled at it. Nevertheless, perhaps biochemists will
find therein reasons for interesting themselves to advantage in biochemical
variation and not solely in the unitarian aspects of biochemistry. Perhaps
chemists will find reasons for recognizing that although it is true that
chemistry' is one, and everywhere obeys the same laws, yet the chemist
within the living cell has his own special methods which can only be
unravelled by means of the experimental study of living material. Perhaps,
also, the essays that follow will assist in convincing certain biologists that
they are wrong when they assert that the natural realities which we des-
cribe by the concepts of species and taxonomic classes no longer exist at
the level of the molecular phenomena which is the study of biochemistry.
In addition, perhaps, they will also become convinced that the field of the
metaboHsm, of cells, of organisms, and of the biosphere itself, offers a
fruitful region for the study of some of the most fundamental problems of
biology.

Although unable to flatter himself with unqualified success in an under-
taking as difficult as this, the author feels confident of the indulgence of
the reader for any omissions or errors he may have committed.

Marcel Florkin

PART ONE

CHAPTER I

THE BIOSPHERE

The terrestrial globe is surrounded by an atmosphere consisting chiefly of
nitrogen, oxygen and argon, but containing other elements and such com-
pounds as carbon dioxide.

The distribution of molecules in the atmosphere is of considerable bio-
chemical interest. At altitudes below 20,000 m the average composition
shown in Table I is maintained constant by convection currents, but at
higher altitudes, due to the difi^erent molecular weights of the gases compos-
ing the atmosphere, a separation by sedimentation becomes apparent.
Beyond 150,000 m, this separation becomes very marked and progres-
sively the atmosphere becomes less dense until finally it fades into the
emptiness of interplanetary space. Nearest to the earth is the troposphere,
its composition being kept more or less constant at the values shown in
Table I, by convection currents. Its thickness varies from 10 to 15,000 m
according to the season and the latitude. Above it is the stratosphere, so-
called because of the stratification of different gases in order of their
molecular weights. The layers of gas are not disturbed by convection
currents and there is no appreciable circulation of molecules in a vertical
direction. Whilst in the troposphere the temperature decreases with alti-
tude, in the stratosphere it is independent of the latter.

At around 80,000 m is the start of the ionosphere which takes its name
from the fact that it is rendered conducting by the ionization phenomena
produced by the sun's ultra-violet radiation. The presence of carbon di-
oxide in the troposphere, as we shall see, is very important despite its low
concentration. The presence in the stratosphere of a diffuse layer of ozone,
the ozonosphere, is not less important for it prevents the greater part of
the sun's ultra-violet radiation from reaching the surface of the earth where
it would otherwise soon put an end to all life.

The earth is also surrounded by a hydrosphere, a discontinuous layer of
water in different physical states which separates the lithosphere from the
atmosphere and extends into the latter in the form of water vapour.

The hydrosphere is made up of oceans, lakes, rivers, streams, water
absorbed by the rocks and by snow and ice. Oceans cover 70% of the
earth's surface and their average depth is 3,800 m, and sea water represents
approximately 98% of the hydrosphere. The composition of the sea
water and its dissolved gases is given in Table II.

1

UNITY AND DIVERSITY IN BIOCHEMISTRY

Table I

Average composition of the troposphere

(from Paneth, completed by Mason, 1952)

Composition

Composition

Total mass

Partial

by volume

by weight

in geograms

pressure

(p.p.m.)

(p.p.m.)

(lO^'Og)

(mmHg)

N,

780,900

755,100

38-648

593-02

o.

209,500

231,500

11-841

159-52

A

9,300

12,800

0-655

7-144

CO2

300

460

0-0233

0-228

Ne

18

12-5

0-000636

He

5-2

0-72

0-000037

CH4

2-2

1-2

0-000062

Kr

1-0

2-9

0-000146

NaO

1-0

1-5

0-000077

H3

0-5

0-03

0-000002

Xe

0-08

0-36

0-000018

Os(^)

0-01

0-36

0-000031

760-00

(1) Variable, increasing with altitude.

Table II

Composition of sea water

(mM per litre)

(after Conway, 1943)

Na

478

K

10

Ca

11

Mg

55

CI

559

SO 4

29

HCO3

2

PO4

traces

Gas dissolved in sea water (ml/l)

(after Mason, 1952)

Oxygen

0-9

Nitrogen

8-4-14-5

Total CO J

34-56

Argon

0-2-0-4

THE BIOSPHERE 3

The total of those parts of the lithosphere, the hydrosphere and the
atmosphere, in which hfe is present, is called the biosphere. What are its
limits ? In the direction of interstellar space it is bounded by the ozono-
sphere, at about 20 km from the ground. Towards the earth's core the
boundar}' is reached \Q.vy soon at the surface of the continents and is not
generally deeper than this by more than 10 m. On the other hand, in the
ocean life can be found at depths down to 10,000 m.

No matter to which theory of the earth's origin one subscribes, it seems
probable that there was a time when the terrestrial atmosphere consisted
almost entirely of nitrogen and carbon dioxide. It is probable that at the
beginning of the earth's history the atmosphere just described had been
rapidly transformed into a mixture of oxygen and nitrogen, for the study
of the rocks does not show the existence of any marked variations, which
shows that the composition of the atmosphere has been much as it is now
throughout a great part of geological time. For instance, the state of oxi-
dation of pre-Cambrian rocks is not significantly different from that of more
recent rocks. The change from an atmosphere of carbon dioxide and
nitrogen to one of oxygen and nitrogen is generally considered to be a
result of the process of photosynthesis.

In the course of time, considered on a geological timescale, the atmos-
phere has had various chemical substances added to it: volcanic gases
(chiefly CO2), oxygen resulting from photosynthesis, COo as a result of
metabolism and the decay of organic material, helium-4 produced by the
radioactivity of uranium and thorium, argon-40 from the decay of potassium-
40. (Natural argon contains the isotopes of mass 36, 38 and 40. Only
argon-40 is derived from potassium-40. Argon-40 constitutes 99-63% of
natural argon. Similarly for helium : only helium-4 is derived from the
a rays emitted by several natural isotopes.)

On the other hand, subtraction of certain chemical substances has taken
place : loss of oxygen by oxidation of iron, sulphur, manganese, etc.,
loss of COo through formation of carbon, petroleum and dead organisms,
loss of CO2 in the formation of carbonates, loss of nitrogen by fixation, loss
of nitrogen by formation of oxides by electrical or photochemical action,
loss of hvdrogen and helium due to the weakness of the earth's
gravitational field, etc.

The equilibrium between losses and gains which has been obtained is
witnessed by the constancy of composition of the atmosphere over extended
periods of geological time. In the special case of carbon dioxide the
regulatory role of the hydrosphere also plays a part.

Certain geochemists consider that the hydrosphere remained shallow
up to the end of the Paleozoic period, so that the oceans as we know them
today have a relatively short histor\\ Others say that the volume of the
oceans has not changed much since the pre-Cambrian period. Whichever

4 UNITY AND DIVERSITY IN BIOCHEMISTRY

is the case it is generally agreed today that the oceans have not changed
appreciably in composition since the Archaean period.

Together with Conway (1943) we may advance four hypotheses to
account for the chemical evolution of the oceans.

1. The water results from the condensation of water vapour from the
primitive atmosphere whilst the chlorides have been added gradually over
the ages (constant volume, volcanic chlorides).

2. Both water and chlorides are the result of an initial condensation
(constant volume, constant chloride).

3. The water and the chlorides have been progressively accumulated
(both oceans and chlorides of volcanic origin).

4. The chlorides were initially present (metallic chlorides in the surface
of the earth), the water being gradually added as a result of volcanic action
(volcanic oceans, constant chloride).

At the present time the third hypothesis is the one favoured by geo-
chemists. According to this view the mass of substances dissolved in the
sea arises from erosion of the terrestrial crust. The total of these substances
is enormous. If all the sea water were evaporated, it would form a layer of
salt 153 m thick covering the surface of the continents. Goldschmidt
calculates that for each litre of sea water 600 g of igneous rock have been
dissolved. In other words, for each square centimetre of the earth's crust,
during the formation of the oceans, erosion has removed 160 kg of igneous
rock. The greater part of this has gone to form the sedimentary rocks.

The biosphere is chronologically after the lithosphere, the hydrosphere
and the atmosphere. Although occupying a portion of all three regions, it is
discontinuous and comprises the total mass of organisms. This definition is
additional to what has already been said about the vital nature of the bio-
sphere, its location and its discontinuous character. The mass of the
biosphere is much less than that of the hydrosphere or the atmosphere.
According to Rankama and Sahama (1950), the relative weights are as
follows :

Hydrosphere 69,100
Atmosphere 300

Biosphere 1

But if the biosphere is quantitatively insignificant, nevertheless it is the
centre of considerable chemical activity and it can be calculated that in the
course of the last 500 million years, that is since the appearance of the
Trilobites, it has "metabolized" a mass of material equal to the total weight
of the globe.

One can obtain an idea of the size of the biosphere by calculating the
annual production of organically bound carbon per square kilometre of the
earth's surface. Riley obtains a figure of 160 metric tons on land and 340

THE BIOSPHERE 5

metric tons in the sea. The total annual production of the continents is
20 ± 5 X W metric tons and that of the sea is 126 ± 82 X 10^ metric
tons, a total of 146 i 83 X 10^ metric tons.

The predominant chemical elements in the biosphere are hydrogen,
carbon, nitrogen, oxygen and phosphorous. Sodium, magnesium, calcium,
potassium, chlorine, sulphur and iron, in addition, are always present in
concentrations ranging from 0-05% to 1%. Further elements, although in
smaller quantity, are always found in measurable amounts : boron, alumin-
ium, copper, zinc, silicon, gallium, molybdenum, manganese, cobalt and
iodine. On occasion other elements may be found in living organisms.

The normal constituents of the biosphere are, with the exception of
iodine, members of the first four periods of the periodic table. These are
the lighter elements. Now, water makes up a large proportion of the
biosphere and it is natural that the elements present in living organisms are
those most widely distributed in the earth's crust and whose derivatives are
most soluble. The electronegativity of the elements also plays an important
part. In a biosphere of a predominantly aqueous nature it is natural that
elements of weak electronegativity forming soluble cations are easily
absorbed and assimilated. The same applies to elements of very strong
electronegativity which give readily soluble anions.

To characterize the quantitative relations existing between the various
elements entering into the composition of living things, one can say that
if one adds up the amounts present of the following eleven metals and
metalloids :

Carbon Sulphur Calcium

Hydrogen Phosphorous Magnesium

Oxygen Chlorine Potassium

Nitrogen Sodium

then one accounts for almost the total weight of the organism— 99-9% in the
case of a man. This is one way of saying that the major part of any organism
is made up of water, lipids, polysaccharides and proteins, and by chlorides,
bicarbonates, phosphates, and sulphates of sodium, potassium, calcium
and magnesium. The elements making up these compounds are the lighter
elements, and, as already stated, are the most common in the surface of
the lithosphere and hydrosphere. They include the elements of very weak
and very strong electronegativity. It is not unexpected that they should
have an important place in the composition of organisms.

As far as the minor elements are concerned we must guard against limit-
ing the Hst too closely. Provided that there is at least one atom per cell of
a trace element, then a function may be assigned to that element. As the
methods of detecting trace elements improve, so does the number of trace
elements increase. As our knowledge of biochemistry increases so does the

6 UNITY AND DIVERSITY IN BIOCHEMISTRY

number of trace elements having a known function increase proportion-
ately. All the more reason, therefore, to treat warily any such idea as that of
an element of biochemical importance.

The lithosphere, the hydrosphere and the biosphere are made up,
qualitatively, of the same elements and only differ in the relative proportions
of these elements.

REFERENCES

Conway, E. J. (1943) The chemical evolution of the ocean. Proc. Roy. Irish Acad.

(B), 48, 161-212.
Leuthardt (1941) MineralstoflFwechsel, Erg. d. Physiol., 44, 588-655.
Mason, B. (1952) Principles of Geochemistry, Wiley, New York.

CHAPTER II

CONSTITUENTS OF THE BIOSPHERE

As LONG as chemistry retained its aura of secrecy and magic ; that is, up to
the seventeenth century, the study of the chemical compounds making up
the biosphere had not even been considered. One may attribute the first
step to Van Helmont who, in his work Ortus medicinae, pubhshed in 1652,
described carbonic anhydride as being present in intestinal gases, separated
an alkaline substance from blood and attempted to resolve human urine
into its constituents. It was necessary to await the development and
elaboration of the idea of chemical compounds before Scheele, in 1775,
succeeded in isolating uric acid from urinary' calculi and PouUetier de la
Salle, in 1782, extracted cholesterol from gall-stones with the aid of alcohol.
Following this came the most spectacular period of organic chemistry
which saw the establishment of the idea of "radicals" (groups of atoms in
the molecule which can be considered as remaining intact throughout a
series of reactions, and which can be transferred from one molecule to
another without undergoing disruption) and the idea of chemical type
(that two substances which possess similar chemical properties have a
similar arrangement of their constituent atoms in the molecule). The
application of methods capable of isolating substances present in living
things without any gross chemical changes taking place resulted in the
identification of a large number of these constituents and the list of these
increases daily. In 1862, Ernest Wagner, professor at Leipzig, in his
Manual of General Pathology, stated an important generalization — that, of
the material of living substances, apart from water, a large percentage can
be divided into three simple organic types which we recognize today as
fatty acids, sugars and amino acids. - A mushroom, a spinach leaf, a sea
urchin egg, the flesh of an oyster, a silk- worm, contain respectively 91*3,
92'3, 77-3, 88*3, and 78-4% of water. The dry residue is made up of these
three principal types of compounds to the extent of 94-2% in the mushroom,
72-6% in the spinach leaf, 93-5% in sea urchin egg, 90-5% in oyster flesh
and 83-9% in the silk- worm. The spinach leaf contains a somewhat smaller
proportion of these organic constituents than the others due to the larger
amounts of inorganic material which amounts to 27-2% of the dry weight.
The sea urchin egg, on the other hand, contains only 1*5% of inorganic
matter, mainly chlorides, sulphates, phosphates and bicarbonates of
sodium, potassium, calcium and magnesium. Nevertheless, as stated above,

8 UNITY AND DIVERSITY IN BIOCHEMISTRY

the biosphere contains small amounts of a great number of other elements
which are used to good effect.

Organic chemistry began by the study of certain natural carbon deriva-
tives. In the course of its spectacular development during the nineteenth
century, it ceased to be a science of naturally occurring compounds and
became a science of imaginary molecules in the sense that a molecule not
present in nature but synthesized by an organic chemist is a product of his
mind and intellect. Nevertheless, a part of organic chemistry forms a
whole segment of the natural science of biochemistry- — this is the organic
chemistry of naturally occurring substances, a field which depends upon the
technical skill of the organic chemist, but whose frontiers and content
primarily interest the biochemist.

This chapter is only a brief outline, and is not a catalogue of the
chemical structures present in the biosphere. In our present state of
knowledge it would certainly be premature to think of establishing such a
catalogue. It is only necessary for an organic chemist to examine minutely
the constituents of such a narrow portion of the biosphere as, for example,
the musk gland of the musk-deer (see E. Lederer; Animal odours and
perfumes, Fortschr. Chem. org. Naturstqffe, 1950, 6, 87-153), toad parotid
gland (V. Deulofeu; The chemistry of the constituents of toad venoms,
Fortschr. Chem. org. Naturstojfe, 1948, 5, 241-266), the cell of a myco-
bacterium (J. Asselineau and E. Lederer; Chimie des lipides bacteriens,
Fortschr. Chem. org. Naturstqffe, 1953, 10, 170-273), or the wood of various
conifers (H. Erdtman; Chemistry of some heartwood constituents of
conifers and their physiological and taxonomic significance. Prog,
org. Chem., 1952, 1, 22-63), for him to obtain an imposing harvest
of new molecules. Many molecules among them are merely variations on a
general theme and their interest is greater still from the point of view of
comparisons between living organisms. At the present time, without
stating categorically that they are present in all organisms, we can make a
list of types of organic structure most widely and generally distributed.

I. THE THREE PRINCIPAL BIOCHEMICAL STRUCTURES

A. Aliphatic Acids

Aliphatic organic acids are very widely distributed in living things,
particularly in the form of fats, hence the name "fatty acids".

The first member of the straight chain saturated series is formic acid
O

H — C corresponding to methane CH4, and the first member

\

OH

CONSTITUENTS OF THE BIOSPHERE

of the unsaturated series is acrylic acid, corresponding to propylene, allyl
alcohol and acraldehyde (acrolein).

CH2

II
CH

I
CH2OH

Allyl alcohol

CH.

II
CH

I

//

O

o

\

OH

Acrylic acid

CH2
II

CH

\

H

Acraldehyde
(Acrolein)

(a) Saturated Fatty Acids

These are all members of a homologous series of general formula
RCOOH, where R represents an aliphatic chain of the type CH3(CH2)a;
or CnH2n+i. The lower members are volatile liquids. Those with six to
nine carbon atoms are oily liquids. Above ten carbon atoms these sub-
stances are solid.

Table III
Natural saturated fatty acids Cn^inOz

Systematic name

Common name

Foiiiiula

M.W.

«-Methanoic

Fonnic

HCOOH

46

n-Ethanoic

Acetic

CH3COOH

60

n-Propanoic

Propionic

C2H5COOH

74

n-Butanoic

Butyric

C3H7COOH

88

«-Pentanoic

Valeric

C4H9COOH

102

«-Hexanoic

Caproic

C5H11COOH

116

n-Octanoic

Caprylic

C7H15COOH

144

n-Decanoic

Capric

C9H19COOH

172

«-Dodecanoic

Laurie

C11H23COOH

200

n-Tetradecanoic

Myristic

C13H27COOH

228

«-Hexadecanoic

Palmitic

C15H31COOH

256

n-Octadecanoic

Stearic

C17H35COOH

284

M-Eicosanoic

Arachidic

C19H39COOH

312

n-Docosanoic

Behenic

C21H43COOH

341

«-Tetracosanoic

Lignoceric

C23H47COOH

369

n-Hexacosanoic

Cerotic

C25H51COOH

397

n-Octacosanoic

Montanic

C27H55COOH

425

n-Triacontanoic

Mellissic

C29H59COOH

453

«-Dotriacontanoic

. . .

C31H63COOH

481

n-Tetratriacontanoic

. . .

C33H67COOH

509

«-Hexatriacontanoic

C35H71COOH

537

n-Octatriacontanoic

...

C37H75COOH

564

10 UNITY AND DIVERSITY IN BIOCHEMISTRY

The lower members are very soluble in water and dissociate weakly ; their
solubility decreases as the number of carbon atoms increases.

Except for formic acid, propionic acid and valeric acid, the saturated
fatty acids generally found in nature are those with an even number of
carbon atoms from 2 to 38. Table III shows their scientific and common
names, their empirical formulae and molecular weights.

{b) Unsaturated Fatty Acids

The unbranched saturated fatty acids form a perfect homologous series
which is represented in nature by the members having an even number of
carbon atoms between 2 and 38 (save for formic, propionic and valeric
acids) but this is not the case with the unsaturated acids. These acids are
made complex by the presence of one or more double bonds which
in turn can undergo several types of reaction such as hydrogenation,
halogenation, etc.

1. One Double Botid

In the same way as the hydrocarbons these acids are named according
to the number of carbon atoms they possess, the carboxyl carbon being
numbered 1. The presence of a double bond is indicated by the suffix -en,
and its position by a numbered prefix, the two numbers indicating which
two carbon atoms are joined by the double bond. Thus the acid C18H34O2,
commonly known as oleic acid, bears the scientific designation 9 : 10-
octadecenoic acid, sometimes abbreviated to 9, octadecenoic acid. It is the
most widely distributed in nature, of the monoethenoic acids, but there are
several others, all with an even number of carbon atoms. The presence of the
double bond admits the existence of a cis and of a trans form. Since nature
only contains the cis forms of the aliphatic acids, in the field of biochemistry
we may consider the prefix cis as understood, and therefore omit it.

2. Tzco or More Double Bonds

Fatty acids having two, three, four, five or six double bonds are not
unusual in nature. Two of these are particularly widely distributed, they
are, linoleic acid, CigHgjOa, or 9:10:12:13-octadecadienoic acid, and
linolenic acid, CigHgoOg or 9:10:12:13:15:16-octadecatrienoic acid.

(c) Branched Chaiji Fatty Acids

Certain fatty acids do not have a linear chain, for example, isovaleric acid,
3-methyl-butanoic acid CH3.CH(CH3).CH2COOH. This is the only
naturally occurring aliphatic acid (besides formic acid, propionic acid and
valeric acid) having an odd number of carbon atoms. Nevertheless, the
main chain in it has an even number. In the unsaturated series examples of
naturally occurring branched chain acids are more common.

CONSTITUENTS OF THE BIOSPHERE

11

(d) Dibasic and Polybasic Acids

These play an important part in the chemistry of Hfe. Present in small
amounts in animal tissues and bacteria they are present in quantity in
vegetable tissues. In both cases they have very important functions.

Only one 1:2 diacid is possible, oxalic acid COOH.COOH, an acid
which is readily oxidized by permanganate to COo and water. The simplest
1:3 diacid is malonic acid COOH.CHg.COOH, which decomposes at
100°C into acetic acid and COo. The 1:4 diacids are represented by
succinic acid COOH.CH2.CH2.COOH. Heat causes their dehydration to
form an internal anhydride.

CH.— COOH

CH,— CO

^ H0O +

CH2— COOH

Succinic acid

\

o

/

CH2— CO

Succinic anhydride

Fumaric acid COOH.CH : CH.COOH, heated at 140X in a closed vessel
with a little water, is changed into its isomer maleic acid.

H— C— COOH H— C— COOH

I! I!

H— C— COOH COOH— CH

Maleic acid Fumaric acid

{e) Hydroxy-acids

Lactic acid CH3CHOH.COOH is one of the acids most commonly
found in nature. When a hydroxy-acid is heated it readily loses water, but
the course of the reaction differs according to the relative positions of the
carboxyl and hydroxyl groups. If the two groups are in the 1, 2 position,
as in lactic acid, thev will from a laciide

R

I
CH

I
COO

OH

H

OOC

R

2H2O + CH— O CO

H

HO

HC

R

CO — O — CH

R

If the carboxyl and hydroxyl groups are in the 1, 3 position then heating
will produce an unsaturated acid :

CH2OH— CH2— COOH

CHo=CH— COOH+HoO

12 UNITY AND DIVERSITY IN BIOCHEMISTRY

If the two functional groups are 1 , 4 or 1 , 5 to each other they will form
an internal ester or a lactone.

CH,— CH2— CH2— CHa CH,— CH,— CHo— CHo

OH COOH O CO

1 . Monohydroxy-monoacids

These substances frequently appear during the process of the breaking
down of cellular nutrients.

The simplest is glycoUic acid CH,OH.COOH.

Lactic acid exists as two position isomers, primary or ^-lactic acid
CH.OH.CHo.COOH, and secondary or a-lactic acid CH3.CHOH.COOH.
This last form possesses an asymmetric carbon atom and therefore exists as
a laevo- and a dextro- rotator}' form.

Nature does not contain hydroxy derivatives of butyric acid other than
j8-hydroxybutyric acid CH3.CHOH.CH2.COOH. Another interesting
monohydroxy-monoacid is hydroxylignoceric acid C22H4;.CHOH.COOH,
an acid found combined in certain cerebrosides.

2. Polyhydroxy-monoacids

The simplest is glyceric acid CH.,OH.CHOH.COOH. IVIany others may
be readily prepared by oxidation of sugars, particularly the aldoses, whose
aldehyde group is more readily oxidizable than the keto group of the ketoses
(see p. 24).

3. Monohydroxy-polyacids

The simplest is tartronic acid COOH. CHOH. COOH, which is a product
of the oxidation of glycerol CHoOH.CHOH.CHoOH, of malonic acid
COOH.CHo.COOH and of tartaric acid.

Two other representatives of this group play an important role in the
biosphere. They are, malic acid COOH. CHOH. CHo.COOH which yields
malonic acid on oxidation and when water is removed is transformed into
maleic and fumaric acids, and citric acid COOH. CH2C(0H). (COOH).
CH2COOH, which is a monohydrox\'-tribasic acid. Citric acid is trans-
formed bv dehvdration at 175° into aconitic acid COOH.CH : C(COOH).
CHo.COOH.

4. Polyhydroxy-polyacids

A series of these compounds is present in the biosphere ; they are derived
from the sugars by oxidation (see p. 24).

CONSTITUENTS OF THE BIOSPHERE

(/) Acid Aldehydes

13

The simplest is glyoxylic acid COOH.CHO, prepared by reduction of
oxalic acid COOH.COOH or oxidation of glycol CHaOH.CHgOH.

Uronic acids are derived from the aldoses by oxidation of the primary
alcohol group to a carboxyl group.

Glucuronic acid is widely distributed in nature, it is the uronic acid
derived from glucose.

H

c=o

HCOH

HOCH

HCOH

I
HCOH

I
COOH

D-glucuronic acid

(g) Keto-acids

The monoketo acids are differentiated by the symbols a, /3, y, S, etc.,
according to the position of the ketone group relative to the carboxyl group.
They can be prepared by oxidizing the corresponding keto-acids or by
hydration of a keto-nitrile. For example, pyruvic acid CH3.CO.COOH is
prepared by hydrating CH3.CO.CN. The most important keto-acids in the
cell are pyruvic, oxalacetic, a-ketoglutaric and oxalosuccinic, and their
decarboxylation is the major source of respiratory carbon dioxide.

O

O

0

0

C COOH

C COOH

C-COOH

C— COOH

CHo

CH,

HC COOH

CH3

COOH

CH2

CH,

COOH

COOH

pyruvic acid

oxalacetic acid

a-ketoglutaric
acid

oxalo-succinic acid

14 UNITY AND DIVERSITY IN BIOCHEMISTRY

The simplest of the jS-keto-acids is acetoacetic acid.

O

//
CH3— C— CH2— c

II \

O OH

There are ^-keto-acids corresponding to the different fatty acids as
shown below :

R— CHo— CH2— COOH -* R— C— CH2— COOH

II

O
The keto-acids can exist in a ketonic or in an enolic form :

R— C— CH2— COOH R— C=CH— COOH

II I

O OH

ketonic form enolic form

B, The Sugars or "Oses"
These are characterized by the presence of the "ose" grouping :

O

c

CHOH

I

The simplest are those with two carbon atoms, the bioses. For example,
glycollic aldehyde, the lowest member of the alcohol-aldehyde series which
is obtained by oxidation of glycol :

H O

\ //

CH2OH 9

CH2OH H-C-H

OH

Glycol Glycollic aldehyde

REFERENCES

Marklen, K. S. (1947) Fatty Acids. Their Chemistry and Physical Properties.

Interscience, New York.
Ralston, A. W. (1948) Fatty Acids and their Derivatives. Wiley, New York.

CONSTITUENTS OF THE BIOSPHERE 15

(a) Aldoses and Ketoses

Sugars are of two types — aldoses, and ketoses. Amongst the C3 sugars,
for example, there is one ketose, dihydroxyacetone, and one aldose, gly-
ceraldehyde. The latter has an asymmetric carbon atom and exists in two
optically active forms.

CH.OH

CHO

(11

CHO

c— 0

H C OH

(3)

HO C H

CH2OH

CH2OH

CH2OH

Dihydroxyacetone

d( + )-Glyceraldehyde

l( — )-GIyceraIdehyde

The two forms are designated D and l and the sign inside the parentheses
indicates the direction of rotation of the plane of polarized light. By using
the procedure of Kiliani (HCN reacts with sugars to form a nitrile-alcohol
which on hydrolysis yields an acid having one carbon atom more than the
starting material. By reduction of this acid a sugar is obtained having one
carbon atom more than the original sugar) one can prepare aldoses higher
up the series by inserting between (C-1) and (C-2), either a group of
configuration

j-j Q OH or of configuration HO — C — H.

I I

In this way one obtains four tetroses, or C4 sugars :

CHO CHO CHO CHO

HO-C-H H— C-OH HO— C— H H— C— OH

1 1 I I

H— C— OH H— C-OH HO— C— H HO— C— H

CH2OH CHoOH CHoOH CH.OH

D-Threose D-Erythrose L-Erythrose L-Threose

The letter d or l preceding the name of the sugar indicates its relation-
ship to one of the parent glyceraldehydes. This is a general rule in sugar
terminology.

R R

I I

H— C— OH HO— C— H

CH.OH CH2OH

n— L—

16

UNITY AND DIVERSITY IN BIOCHEMISTRY

0 X I X I

1 o o o o

I X I ..

CO

<u

I

Q

S

fj

o

"Tr^

"«^

>

■^

l-H

^

1-1
W

S^

<

a

H

a

K

1-i

CO

CO

V

'u
u

CO

I

1-1

O

O I X

X o o
u-u-o ■

X ..
X

0 I I I

1 o o o

■ u-u-u— u-

X X ~

O XXI

X X o o o
u-u-u- u-u-u

O I I ^.

X I

0 X I I I I

1 O C 0 o o
u-u-u-u-u-u

I I I I -

HSOnV-a

O X I X X

X I o o o o
u-u-u- u-u-u

O X X X ^.

Hso-anv-n

CI XII
I O X O O 0

u-u-y-u-u-u

X ^ X X ^.

3SODmO-o

0 III

1 I I o o o
u-u-u-y-u-u

I I "^ ^ I

3SONNVl\-o

0 XI

1 I o o
u- u-u-u

0 X -

1 X

0 I II

1 O I O O

-u-u-u-u-u

I O X -.

I I

0 II

1 I I o o

U-U-U-U-U-U
O O X -,

XX I

HSOmO-a

jsoai-a

3SO±DVlVO-o

3S01VX-a

Oil I

I O O X o

r-u— u— u— u-u

X X O r.

X X

M
u

O I

X I O

• u-^-u-

X X

ex X
X O X o
-u-u-u— u-
X o -

X I

o

I I

O I

J u-u-y-u-u-u
O X o -.

X XI

^ I 1_

5 t

u-u-
I o

I

X X

u-u-
O o

X X

GIG
u-u-u

X O ^.

I I

I X

q I o
-u-u-u

X O „

X I

OX I

X O X X O

i->u-u-y-u-u
X 5 o ~

XXI

0 I

1 I I O
.u-^-c^-u-

I I X

X
X I O

■u-u-u

O O -,

III

3S01V1-T

3S01DV1V0-T

3Soai-T

3Soino-T

3SONNVN-T

OX I
I I 0 I I 0
u-u-u-u-u-u
G X 2 o ^^

3S03mD-T

2 § X X X §

U-u-U-^-^-U
X I I X

3SO\inv-i

HOD'H
HDOH
HDOH
HDOH
HDOH

OHD

3Sonv-i

CONSTITUENTS OF THE BIOSPHERE

17

Table V

Ketoses {natural

and synthetic)

CHoOH

CHoOH

1

CH2OH

1

c=o

1

1

c=o

CHoOH

1

1

c=o

HO-C-H

H-C-OH

1

1

HO-C-H

1

H-C-OH

1
HO-C-H

CHoOH

CH2OH

CHoOH

CH2OH

Dihydroxy-

L-Erythrulose

D-Xyloketose

L-Xyloketose

acetone

D-Xylulose

L-Xylulose

CH2OH

CH2OH

CH2OH

CH2OH

c=o

c=o

c=o

c=o

HO— C— H

H-C-OH

H— C-OH

HO-C-H

H-C— OH

HO-C-H

H— C— OH

HO— C-H

H-C-OH

HO-C-H

CH2OH

CHoOH

1

CH2OH

CH2OH

D-Riboketose

L-Riboketose

D-Ribulose

L-Ribulose

D- Fructose

L-Fructose

CHoOH

CHoOH

CH2OH

CH2OH

c=o

c=o

1

c=o

c=o

H-C-OH

1
HO-C-H

HO— C-H

HO-C-H

HO-C-H

1

H-C-OH

HO-C-H

HO-C— H

1

1
H-C-OH

HO— C— H

1

H— C— OH

HO-C-H

CH2OH

1
CH2OH

CH2OH

CH2OH

D-Sorbose

L-Sorbose

D-Tagatose

L-Psicose

18 UNITY AND DIVERSITY IN BIOCHEMISTRY

Table V {continued)

CHoOH CH2OH CH2OH CH2OH CH2OH

I ' I I I I

C=0 C=0 C=:0 c=o c=o

I I I I i

HO— C— H HO— C— H HO— C— H H— C— OH HO— C— H

I I I I I

H-C— OH HO— C— H H— C— OH HO— C— H H— C-OH

i I I I !

H— C— OH K— C— OH H— C— OH H— C— OH HO— C— H

I I I I I

H— C— OH H— C— OH HO— C— H H— C— OH HO— C— H

I i I I !

CH2OH CH2OH CHoOH CH2OH CHoOH

D-Sedoheptose D-Manno- L-Perseulose D-Gluco- L-GIuco-

D-Sedoheptulose ketoheptose L-Galaheptulose ketoheptose ketoheptose

D-Manno-

heptulose

The direction of rotation is indicated by (+) or (— ), as in the following
examples : d(— ) ribose, d(+) glucose, l(— ) glucose, d(— ) arabinose.

From the four tetroses, by further syntheses it is possible to obtain eight
pentoses, which will be aldopentoses, and from these, sixteen aldohexoses
(Table IV). The configuration of the ketoses can be deduced from that of
the aldoses, according to whether they derive from D-glyceraldehyde or
from L-glyceraldehyde. Table V shows a series of known ketoses, both
natural and synthetic.

(b) Cyclic Formulae

In the above, the sugars have been considered as straight chain molecules,
but it has been established that in the crystals of sugars containing more
than 4 carbon atoms, cyclic tautomeric forms are present. In solution these
sugars exist almost completely as cyclic molecules in equilibrium with small
amounts of the straight chain form.

The fact that aldoses having more than 4 carbon atoms do not give certain
reactions given by aldehydes is due to their being predominantly in the
cyclic form. On the other hand, when a solution of glucose is prepared, the
rotatory power of the solution progressively decreases until it reaches a
certain value at which it remains constant (mutarotation). This is due to
the fact that glucose exists in the form of two cyclic isomers, and that an
equilibrium is set up between these two isomers and small amounts of the
straight chain form, as shown in the formulae below :

THE CONSTITUENTS OF BIOSPHERE

19

HO H

\ /

C

H— C— OH

I
HO— C— H

I
H— C— OH

H— C

O

CH.OH

j3-D-glucose

H— C=0

H— C— OH

I
HO— C— H

H— C— OH

H— C— OH

I
CH.OH

D-glucose chain molecules
(traces)

H OH

\ /
C

H— C— OH
HO— C— H
H— C— OH
H— C

O

CHoOH

a-D-glucose

In the above formulae the ring is made up of five carbon atoms and an
oxygen atom, but in addition the presence has been demonstrated in
solutions of the sugars of molecules in which the ring is made up of four
carbon atoms and one oxygen atom, and derivatives of these forms have
been prepared. Because these rings correspond to those of furan and pyran,
Haworth has proposed a terminolog}^ in which the sugars are considered as
derivatives of these structures.

O

/\
HC CH

II II

HC CH

\/
C

Pyran

o

/ \

HC CH

\ //

c— c

H H

Furan

The pyranose and furanose formulae are drawn in perspective looking
down on the ring, each of the corners representing a carbon atom.

The atoms whose symbols are written above the ring are situated above
the plane of the ring and those whose symbols are written below are
situated below the plane of the ring. These formulae contain an asymmetric
carbon atom additional to those present in the linear formulae and this is
the one which, in D-glucopyranose, for example, is in position 1.

20

UNITY AND DIVERSITY IN BIOCHEMISTRY

HO

(6)
CH.OH

HO

a form

HO

D-glucopyranose

CH„OH

OH

j8 form

Although they are both dextrorotatory, the specific rotations of the two
forms of D-glucopyranose are different.

The unstable form of glucose is glucofuranose. This compound also
exists in the two forms a and /3.

HOH,C — HOHC

'(«) (n

H ^(«)

OH HOHj C — HOHC

HO

OH

HO

D-glucofuranose

One can prepare crystals of the a form or of the j8 form of D-gluco-
pyranose. The first will be obtained by crystallization from aqueous
solution, and the second by crystallization from pyridine. The two forms
are distinguishable by a number of properties, as, for example, their melting
point and their specific rotation in a freshly made solution. In solution,
one form changes into the other, so that the rotation changes until an
equilibrium is reached (mutarotation equilibrium). In the formulae of
Haworth, the keto and aldehyde groupings are not written as in the linear
formula, they are replaced by a potential-aldehyde or a potential-ketone
group.

Ov OH

■Ov H

Potential aldehyde group

Oy^ CH^jOH

"^ OH

OH

CHoOH

Potential ketone group

The sugars are in effect reducing agents, the same as the aldehydes and
ketones, but they do not restore the colour to fuchsin bleached by SOg.

Haworth's formulae are now generally accepted. They give a clearer idea
of the relationship of the various groups than the ordinary cyclic formulae
or the linear formulae, although any of the three may be met with.

CONSTITUENTS OF THE BIOSPHERE

21

Haworth's formulae take into account certain properties not immediately
apparent when the other formulae are used. For example, the a form of

H OH

\ /
C

H— C— OH

HO— C— H

H— C— OH

H— C

O

CHoOH

a-D-glucopyranose

H OH

\ /
C

H— C— OH

HO— C— H

H— C

H— C— OH

O

CH2OH

a-D-glucofuranose

D-glucopyranose when heated yields an anhydride, a glucosan formed by
elimination of water between the hydroxyl of C-1 and the hydroxyl of
C-2:

CH.OH

O H

OH "N... y^ OH

H OH

^-D-glucopyranose

CH2OH

glucosan

On the other hand /S-D-glucopyranose, under the same conditions, gives a
different anhydride, levoglucosan, resulting from the elimination of water
between the hydroxyl of C-1 and that of C-6, which is explicable from the
closeness of the two carbons in the formulae of Haworth.

CH2OH

H OH

j8-D-glucopyranose

H OH

levoglucosan

22 UNITY AND DIVERSITY IN BIOCHEMISTRY

(c) Natural Sugars

One finds in Nature a selection of sugars, either free or combined. The
lower members of this group are a diose, glycollic aldehyde; two trioses,
glyceraldehyde and dihydroxy-acetone, and a tetrose, D-erythrose. The
pentoses of the biosphere which have been found up to the present are
D-xylose, D-ribose, D-ribulose, D-arabinose and L-arabinose. Among the
hexoses the commonest is D-glucose. Other aldohexoses in the biosphere
are D-mannose, D-galactose and L-galactose. The ketohexose D-fructose is
also very common. Two C7 sugars, sedoheptulose and D-mannoheptulose
have also been identified. It is clear that the natural sugars are most often
members of the D-series.

(d) Amino Sugars

Amongst the large number of sjmthetic compounds two are also of
natural origin

H— C==0 H— C=0

H— C— NH, H— C— NH2

HO— C— H HO— C— H

i I

H— C— OH HO— C— H

H— C— OH H— C— OH

CH2OH CH2OH

Glucosamine Galactosamine

(2-desoxy-2-amino-P-D-glucose) (2-desoxy-2-amino-p-D-galactose)

These are aldohexoses aminated in position 2.

Glucosamine is a constituent of many polysaccharides and mucopoly-
saccharides, and of chitin. Galactosamine is a constituent of cartilage.

(e) Desoxy-Sugars or "Desoses"

These are the result of the removal of an oxygen atom from a hydroxyl
group. There are a certain number of natural desoses, the principal ones
being L-rhamnose, L-fucose and 2-desoxyribose or ribodesose. The
desoses give most of the reactions of sugars. They are unstable.

CONSTITUENTS OF THE BIOSPHERE
H-C=0 H— C=0

23

HCOH

i
HCOH

HOCH

HOCH

CH3

L-rhamnose

(6-desoxy-L-mannose)

HOCH
HCOH
HCOH

HOCH

CH3

L-fucose
(6-desoxy-L-galactose)

H — C=:0

HCH
HCOH
HCOH
CH2OH

2-desoxyribose

(/) Polyhydroxy Compounds Related to Sugars
The reduction of ketoses and aldoses produces the corresponding
alcohols. The reduction of glyceraldehyde gives glycerol, ribitol arises from
ribose and sorbitol is formed from glucose.

CH.OH

CHoOH

H— C— OH

H C OH

HO C H

CHoOH

H C OH

H C OH

CHOH

H C OH

H C OH

CH2OH CHoOH CHoOH

Glycerol Ribitol D-sorbitol

The carbocylic alcohols like inositol are similar types of polyalcohol.

OH
C

H^

HOCH

HCOH

HOCH

HCOH

.H,
C
OH

Inositol

24

UNITY AND DIVERSITY IN BIOCHEMISTRY

(g) Oxidation Products of Sugars
{Monoacidic Polyalcohols, or Polyacids)

Under controlled conditions oxidation of aldoses can give rise either to
aldonic acids resulting from oxidation of the aldehyde group to a carboxyl
group, or to uronic acids in which the aldehyde group is preserved and the
primary hydroxyl is oxidized to carboxyl, or finally to dicarboxylic acids
having a carboxyl at each end. In the case of glucose the following com-
pounds are obtained :

CHO

!

(CHOH)4
CHoOH

Glucose
(aldose)

COOH

(CHOH)4
CHoOH

Gluconic acid
(hexonic acid)

CHO

(CHOH)4
COOH

Glucuronic acid
(uronic acid)

COOH

(CHOH)4
COOH

Saccharic acid
(dicarboxylic acid)

Numerous hexonic, uronic and dicarboxylic acids derived from the
aldoses are present in nature.

C. Amino Acids

{a) Definition

The amino acids are defined as substances having a carboxyl and an
amino group together in the same molecule. The number of substances
falling into this category which organic chemists can synthesize is very
great but only a limited number of these compounds are present in the
biosphere. The amino acids usually have the amino group in a position a to
the carboxyl group, so that their general formula is :

NHa

R— CH— COOH

REFERENCES

HoNEYMAN, J. (1948) An Introduction to the Chemistry of Carbohydrates. Clarendon

Press, Oxford.
PiGMAN, W. W. & GoEPP, R. M. (1948) Chemistry of the Carbohydrates. Academic

Press, New York.

CONSTITUENTS OF THE BIOSPHERE 25

(b) Amino Acids Universally Distributed in Organic Materials

In all living things which have been studied in this connection, one finds
the twenty amino acids listed in Table VI. The natural amino acids can
be considered as derivatives of L-serine, related to L-glyceraldehyde.

NH2 OH

CH2OH— C— COOH CH.OH— C— COH

H H

L-serine L-glyceraldehyde

(c) Amino Acids Occasionally Found

Besides the twenty amino acids which are universally distributed in the
living organism one finds, here and there, certain special amino acids.
Among these are the following :

Hydroxylysine, a, e-diamino-S-hydroxycaproic acid

NH2

H2N— CH2— CHOH— CH2— CH2— CH— COOH

Ornithine, a, S-diamino- valeric acid

NH2

H2N— CH2— CHo— CH2— CH— COOH

^-alanine, /3-amino-propionic acid

CH2— CH2— COOH

NH2

CitruUine, a-amino-carbamidovaleric acid

NH2

H2N— C— NH— CH2— CH2— CH2— C— COOH

y-amino-butyric acid

H2N—CH2—CH,— CHo— COOH

and several others : octopine, a-aminobutyric acid, djenkolic acid, ^-
thiolvaline, canavanine, etc.

26 UNITY AND DIVERSITY IN BIOCHEMISTRY

Table VI
NH2

a-amino acids |

(R-CH— COOH)

(Symbols indicating the nature of the radical R : aliphatic (Al), aromatic (Ar),
heterocyclic (He), acidic (A), basic (B), neutral (N), polar (P), feebly polar (FP),
non-polar (NP), ionic (I), feebly ionic (FI), non-ionic (NI).)

L (+) Alanine (Ala), a-aminopropionic acid

NH2

R = methyl (Al, N, NP, NI) CH3-CH-COOH

L (+) Arginine (Arg), a-amino-S-guanido-w-valeric acid

R = tu-guanido-n-propyl (Al, B, P, I)

NHa

5 Y P «l

CH2-CH2-CH2-CH-COOH

I
NH

/

C=NH

\

NH2

L ( — ) Aspartic acid (Asp), a-aminosuccinic acid

NH2

R = carboxymethyl (Al, A, P, I) HOOC— CH2— CH— COOH

L (— ) Cysteine (CySH), a-amino-/3-thiolpropionic acid

NH2

I
R = thiomethyl (Al, N, P, FI) HS-CH2— CH-COOH

L (— ) Cystine (CyS), j3, jS'-dithio-bis (a-aminopropionic acid)

NH2

R = dimethyldisulphide (Al, N, NP, NI) S-CH2-CH-COOH

NH2
S— CH2-CH— COOH

L (-|-) Glutamic acid (Glu), a-aminoglutaric acid

NH2

I
R = carboxyethyl (Al, A, P, I) HOOC— CHo—CHo—CH— COOH

CONSTITUENTS OF THE BIOSPHERE 27

Table VI {continued)
Glycine (Gly), aminoacetic acid

R = H - (Al, N, NP, NI) H-CK-CCOH

NHq
L (— ) Histidine (His), a-amino-jS(5)-imidazolyIpropionic acid

I
R = imidazolylmethyl (He, B, P, I) N C-CH2— CH— COOH

li 11

HC CH

\ /

N

H
L ( — ) Hydroxyproline (Hypro), 4-hydroxypyrrolidine-2-carboxylic acid

H

R = - (He, N, P, NI) HO— C CH;

1 I

HX C— COOH

\ /H
N
H
L ( + ) Isoleucine, a-amino-j8-methyl-n-vaIeric acid

R = a-methyl-«-propyl (Al, N, NP, NI)

NH2

CH3— CH2— CH— CH-COOH

CH3
L ( — ) Leucine (Leu), a-amino-isocaproic acid

R = isobutyKAl, N, NP, NI) CH3 NH2

\ 1

CH—CH2— CH-COOH

/

CH3
L (+) Lysine (Lys), a-e-diamino-n-caproic acid

R ^ co-amino-n-butvl (Al, B, P, I)

NH2
e 6 Y |3 aj

CH2—CH2—CH2—CH2— CH-COOH

I
NH2

L (— ) Methionine (Met), a-amino-methylthiol-w-butyric acid

NH.

I
R = methylthioethyl (Al, N, P, NI) CHa-S-CHo-CH.-CH-COOH

28 UNITY AND DIVERSITY IN BIOCHEMISTRY

Table VI {continued)

L (+) Phenylalanine (Phe), a-amino-jS-phenylpropionic acid

R = benzyl (Ar, N, NP, NI)

NHa

CH,-CH-COOH

L (— ) Proline (Pro), pyrrolidine-2-carboxylic acid

R = — (He, N, NP, NI) HoC CHj

I I

H2C C— COOH

\ /H

N
H

L (— ) Serine (Ser), a-amino-j3-hydroxypropionic acid

NH2

I •
R = hydroxymethyl (Al, N, P, NI) HO— CH2— CH— COOH

L (— ) Threonine (Thr), a-amino-jS-hydroxybutyric acid

NH2

I
R = hydroxyethyl (Al. N. P, NI) CH3-CH-CH-COOH

OH

L (— ) = Tryptophan'(Try), a-amino-j8(3)-indolepropionic acid

NH2

I

/\ n -CHa-CH-COOH

P a

R = skatolyl (He, Ar, N, NP, NI)

NA.

H

L (— ) Tyrosine (Tyr), a-amino-^(p-hydroxyphenyl) propionic acid
R = /)-hydroxybenzyl (Ar, N, FP, FI)

NH2

HO-C >-CH2-CH-COOH

L (— ) Valine (Val), a-aminoisovaleric acid CH3 NHo

\ I

CH— CH-COOH

R = isopropyl (Al, N, NP, NI) ^{^

CH3

CONSTITUENTS OF THE BIOSPHERE

29

There are also many iminoacids, such as pipecolic acid, baikiaine,
guvacine, etc.

C

/ \
H2C CH:,

H
C

// \
HC CH2

H2C

CH— COOH

H2C

CH— COOH

\ /

N
H

Pipecolic acid

\ /

N
H

Baikiaine

H
C

/ ^
HjC C— COOH

HoC CH2

\ /

N
H

Guvacine

(d) Stereoisomerism
Whilst the natural sugars are for the most part members of the D-series,
the natural amino acids, regardless of their optical activity, belong as a rule
to the L-series,

Natural L-form

D-form

In addition there are certain amino acids which, in addition to the a
carbon atom, contain a second asymmetric carbon (threonine, hydroxy-
proline, isoleucine, hydroxy lysine). These amino acids can theoretically
exist in four isomeric forms.

30 UNITY AND DIVERSITY IN BIOCHEMISTRY

For example in the case of threonine, the four stereoisomers would have
the following formulae :

COOH COOH COOH COOH

ijNCH

HCNH2

H.NCH

HCNH,

HCOH

HOCH

HOCH

HCOH

CHa

CH,

CH3

CHh

L-threonine

D-threonine

L-allothreonine

D-allothreonine

The designation L- is reserved for the form found present in proteins, its
isomer having opposite configurations on carbon atoms a and j8, is distin-
guished by the prefix D-. The other isomers, which also form a pair of
optical isomers, but which are diastereoisomers, are denoted by the prefix
alio, as shown in the diagram for threonine. (In the case of cystine, there
are also two asymmetric carbon atoms, but since cystine is formed by the
union of two molecules of cysteine, then, as in tartaric acid, there are only
three isomers : ll, dd and meso, the meso form being optically inactive.)

Although it is true, in general, that the natural amino acids are a-amino
acids of the l- series, it sometimes happens that in organisms one encoun-
ters stereoisomers of the d- series, for example, D-^S-thiolvaline, D-leucine,
D-alanine, etc.

II. OTHER CHEMICAL STRUCTURES OF GENERAL INTEREST

A. Terpenes

The terpenes and their derivatives are a group of natural substances
related to isoprene CgHg.

CH3

I
CH2=C— CH=CH2

Isoprene

Isoprene does not exist as such in nature, but it is found in the biosphere
in the form of numerous polyisoprenes. These substances may be divided
into three groups : the lower terpenes, the carotenoids and the polyterpenes.

REFERENCES

DESNtTELLE, P. : The general chemistry of amino acids and peptides, in H. Neurath

& K. Bailey, The Proteins, Vol. I, part A, 87-180.
Dunn, M. S. & Rockland, L. B. (1947) The preparation and criteria of purity of
the amino acids. Advanc. Protein Chem. 3, 295-382.

CONSTITUENTS OF THE BIOSPHERE 31

(a) Lower Terpenes
These result from the condensation of two (monoterpenes), three
(sesquiterpenes), four (diterpenes), six (triterpenes), or eight (tetraterpenes)
isoprene residues. The lower terpenes are widely distributed in the essen-
tial oils of many plants. Phytol, an unsaturated alcohol present in the
chlorophyll molecule, is a diterpene derivative.

CH3— CH— (CH2) 3— CH— (CH2) 3— CH— (CH., ) 3— C=CH— CH2OH

CH3 CH3 CH3 CH3

Phytol

(6) Carotenoids
1. Definition

One of the groups of natural isoprene derivatives is that of the caroten-
oids— aliphatic or alicyclic pigments whose colours range from yellow to
red. According to Karrer, they are derivatives of several isoprene units
(often eight) joined together in a manner such that the two methyl groups
at the centre of the molecule are 1 : 6 to each other, whilst the remaining
lateral methyl groups are in the 1 : 5 positions.

— C=CH-CH=CH-C=CH— CH=CH-CH=C-CH-CH-CH=C-
I I

CH3 CH3

I

I

©- <p

CH, CH,

I

(j> <D

O ' -d)

The chain of conjugated double bonds forms the chromophore system
of the carotenoid pigments.

2. Classification

There are two classes of natural carotenoids : the carotenoids proper,
having a C-40 chain (8 isoprene residues), and the carotenoids possessing
less than 40 carbon atoms. The natural carotenoids can be considered as
related to lycopene (C40H56), the red pigment of the tomato.

CHa CH3 H.C CH,

\ / \ /

Ci CH, CH» CH, CH, rC

// (< 1 \ I 1 I 7' 6' \

2CH CHCHzrCHC=CHCH=CHC=CHCH=CHCH=CCH = CHCH=iCCH=CHCH 2'CH

! !| 8 9 10 II 12 13 14 15 15' 14' 13' 12' 11' 10' 9'8' II I

3.CH2 C C 3'CH,

\ /S\ /S'\ /

\ / CH, HaC CHc

CHj Lycopene 4'

4

32 UNITY AND DIVERSITY IN BIOCHEMISTRY

Table VII
Natural carotenoids whose structure is known

(Karrer and Jucker)

CHa CHs CH« CHs

\ / \ /

C CHo CHa CHa CHs C

/ I I I I \

CH CHCH = CHC=CHCH = CHC=:CHCH = CHCH=C-CH=CHCH = C-CH=:CHCH CH

I II II I

CH, CCH, H,CC CH,

\ y I.ycopene \ /

CH, CHi

CH, CH, CH, CHg

\ / \ /

C CH, CH; CHs CHa C

/ I I I I / \

CH CHCH=:CHC=CHCH = CHC=CHCH = CHCH=CCH=CHCHrrCCH = CHC CH,

I II II I

CH, CCH, H.CC CH.

X y Y-Carotene \ y

CH, CH,

CHi CHj CHj CH»

\ / \ /

C CHi CH. CH, CH, C

/ \ I I I I / \

CH, CCH = CH-C = CHCH = CHC = CHCH = CHCH=CCH=:CHCH=CCH=CHC CH,

I II ' II I

CH, CCH, H,CC CH,

\ / B-Carotene \ /

CH, CH,

CH, CH. CH, CHs

\ / \ /

C CH, CH, CH. CHa C

/ \ I I I I / \

CH, CCH = CHC=CHCH = CHC = CHCH=zCHCH = C CH = CHCH=C CH = CHCH CH:

I II II

CH, CCH, H,C C CHj

\ / a-Carolene ^ /

CH, CH

CH, CH. "^

\ /
, V CHs CH»

CH, C:=CHCH, CH3 CH, CH, ^C^

III! I I I / \

CH, C CH C=CHCH = CHC=CHCH = CHCH = C CH = CHCH = C CHrrCHC CH,

\ / \ / II I

^"' u° "^C C CH,

CH. \ /

CH,

Mutatochrome, Citroxanthin

CONSTITUENTS OF THE BIOSPHERE 33

CHa CH, CH. CHj

\ / \ /

C CH3 CH^ CHa CHj C

/ \ I I I I / \

CH, CCHi^CH C=CHCH = CHC = CHCH=:CHCH=:C CH = CHCH=C CH = CHCH CH,

I |\ II

O H,CC CH,

11/ \ /

CH. C CH

\ / \
CH, CH,

a-Carotenepoxide

CH, CH, Cri3 CH,

\ / \ /

C CH= CH, CHs CH, C

/ 1 I I I \

CH CHCH=:CH C = CHCH=CHC=CHCH=CHCH=CCH=:CHCH = CCH=CHCH CH

I il II I

CH, CCH. HjCC CHOH

\ / ^ -^^

CHa • ^"'

Lycoxanthin

CHj CHs CHi CHj

\ / \ /

C CH, CH, CH, CH, C

/ I I I I \

CH CHCH = CHC=CHCH=:CH C = CHCH = CHCH = C CHr=CHCH = CCH = CHCH CH

I !! I! I

HOCH CCHa H,CC CHOH

\ / \ /

CH, CH.

Lycophyll

CH. CR,

\ /
CHs CH, CH, CH, CH

! • I II \

CHo CHCHnCHC=CHCH = CHC = CHCH = CHCH = CCH = CHCH = C CH = CHCH CH,

II I

H,CC CGCHi

\ /
CH

Rhodcviolascin (?)

CH, CH, CH, CH,

\ / \ /

C CH, CH, CH, CH. C

/ \ I I I ' \

CH2 CCH=:CHC=CHCH=:CHC=iCHCH=rCHCH = C CH = CHCH=CCH = CH CH CH

II II I

HOCH CCHa H,C C CH,

\ / \ /

CH. CHj

Rubixanthin

D

CH3

CH,

\

/

CH

/

CH,

1

CHCl

H,COC

!
CCH,

\

/

CH

34 UNITY AND DIVERSITY IN BIOCHEMISTRY

Table VII (continued)

CH, CHa

\ /

C CH, CH,

/ \ \ /

CH, C CH CH, CH, CHa CH, C

I I I I I I I \

HOCH C CHC = CHCH = CHC = CHCH=CHCH = CCH=CHCH = C CHz=CHCH CH

\ /l\ / Hi

CH,| O H3CC CH,

CHa Rubichrome \ /

CH.

CH, CHa CH, CPL

\ / \ /

C CH, CH, CH, CHa C

/ \ I I I I / \

CHj C CH=:CH- C = CHCHrrCH C = CHCH = CHCH = C CH=CHCH=CCH=CHC CH,

I li II I

CH: C CH, HaCC CHOH

\ / Cryptoxaiitlun \ /

CH~ CH,

CH, CH, CH, CH.

\ / \ /

C CH, CH, CH. CH, CH

/ \ I I I I \

CH, CCH=CH C = CHCH=:CH C = CHCH=CHCH=CCHrrCHCH = C CH=CHCH CH

I II II II

CH, C CH, H,CC CH

V / Myxoxanthin \ y

CH, CO

CHa CHs CHs CHs

\ / \ /

C CH, CH, CHa CH, C

/ \ I I I I / \

CH, CCH=CHC = CHCH = CH C = CHCH=CHCH=CCH = CHCH = CCH=CHC CH.

I II II I

CH, C CH, HaCC CO

\ / Aphanlne (V) \ y

CH. CH,

CH, CH, CH, CH,

\ / \ /

C CH, CH, CH, CH, C

/ \ I I I I / \

CH, CCH = CH C = CHCH = CHC = CHCH=CHCH=:CCH=CHCH:rCCH=CHCH CH,

I II II I

HOCH C CH, H,CC CHOH

\ / Zeaxanthin \ /

CH, CH,

CH, CH, CHa CH,

\ / \ /

C CH, CH, CH» CHa C

/ \ I I I I / \

CH. CCH = CH C = CHCH=:CH Cr:CHCH=CHCH = CCH = CHCH=C- CH = CHCH CH,

I II II

HOCH C CH, H3CC CHOH

\ / Xanthophyll \ /

CH. CH

CONSTITUENTS OF THE BIOSPHERE

35

CHs CHa CH, CH,

\ / \ /

C CH, CHa CH3 ClU C

/ \ I I I I / \

CHj CCH = CHC=CHCH = CHC=CHCHrrCHCH = CCH = CHCH = CCH = CHC CH^

O H,,CC CHOH

/ \ /

HOCH C CH,

\ / \
CH, CH,

Antheraxaiithin

CH3 CHs CHs CH»

\ / \ /

C CH3 CH3 CH3 CH3 c

/ \ t I I I / \

CHa CCH=:CHC=CHCH=CHC=CHCH=CHCH = CCH = CHCH = CCH=CH-CH CHz

\

O

HOCH C

\ / \
CH, CH,

Xanthophyllepoxide

I
HaCC CHOH

\ /
CH

CHs CH,

\ /
C

/ \

CH, C =

CH3 CH,

\ /
:CH CH, CH3 CH, CH, C

1 I I I 1 I I / \

HOCH C CHC=CHCH=CHC=CHCHrrCHCH = CCH = CHCH= CCH=CHCH CH,

CH,

\

O

CH,

Flavoxanthin, Chrysanthemaxanthin

I I

C CHOH

/ \ /
H3C CH

CHt CH, CHs CH,

\ / \ /

C CHs CHa CHs CH, C

/ \ I I I I / \

CH, CCH=CHC=:CHCH=CH-C=CHCH=:CHCH=CCH = CHCH= CCH=rCHC CH,

\ /

O O

HOCH C/ \

\ / \ C CHOH

CH, CH, ViolaxanthiP / \ /

H,C CH,

CH, CH,

\ /
C

/ \

CH,

=CH CHs

CHs

CHs

CHs CH,
\ /

C
/ \
HsC CH==C CH,

HOCH

CH,

\ /
O
CH,

CHC=CHCH=CH-C=CHCH = CHCH=C-CH=CHCH=CCH

CHOH

Auroxanthin

o

H.C

\ /
CH,

36 UNITY AND DIVERSITY IN BIOCHEMISTRY

Table VII (continued)

CH, CH, CH3 CH.

\ / \ /

C CH, CH, CH, CH, C

/ \ I \' I I / \

CH2 C = CHCHrrCCH = CHCH=:CCH = CHCH=CHC=CHCH=CH- C=CHCH = C CH,

II II

CO CCH. H3CC CO

\ ^ Rhodoxanthin ^ /

CH CH

CHi CH, CH5 CH,

\ / \ /

C CH, CH^ CH, CH, C

/ \ I I I I / \

CHj CCHrrCH C = CHCH = CH C = CHCH = CHCH= C CH = CHCH::rCCH = CH CO CH,

I II I

HOCH C CH, H,C CHs CHOH

\ / Cnpsanthin \ /

CH, CH,

CHa CH, CH, CH,

\ / \ /

C CH, CH, CH, CH, C

/ \ I I 1 I / \

CH. CO CH = CH- C = CHCH = CH C = CHCH = CHCH= CCH = CHCH-CCH=CHCO CH,

I I

HOCH CH,CH, H,C CH, CHOH

\ / Capsorubin \ /

CH, CH,

CH, CH, CH, CH,

\ / \ /

C CH, CH, CH, CH, C

/ \ I I I I / \

CH, C CH^CH C=CHCH = CHC = CHCH = CHCH:r:CCH=CHCH=CCH=CHC CH,

I il II I

HOCH C CH, HaCC CHOH

\ y/ A.staxaiithin \^. y

CO CO

CH, CH, CHa CH,

\ / \ /

C CH, CH, CH, CH, C

/ \ I I I 1 / \

CH, CCH=CHC=CHCH = CHC=CHCH:z:CHCHr=CCHrrCHCH= CCH = CHC CH,

I II II I

CO C-CH, Astacine HsCC CO

\ / • \ /

CO CO

CH* CH,

\ /

C CHa CH, CH, CH,

/ \ I I I I

CH, CCH=CHC=CHCH=CHC=CHCH:=CHCH=CCHzzCHCH=CCH=:CHCH COOH

I II II I

CH, CCH, HaCC CH

\ / Torularhodim (?) \ /^

CH, CH

CONSTITUENTS OF THE BIOSPHERE 37

CH, CH,

\ /

C OH CH3 CH, CHi

/ \ / \ I I

CHj CCH=CHC=zCHCH = CHC=CHCH = CHCH=C CHzrCHCOOH

I I

CHa CCH,
\ / \ Azafrine

CHa OH

CH, CH,
\ /

C CH, CH, CH, CH,

/ \ I I I I

CH, CCH=CHC=CHCH=CHC=CHCHr=CHCH = CCH=CHCH = CCHO

I II

HOCH CCH,

\ / ^Citiaui-ine

CH,

CHi CH, CH, CH,

11 II

HOOCC=CHCH=CHC=CHCH=CHCH=CCH=CHCH = CCOOH

Crocetin

CH, CH, CH, CH,

II II

HOOCCH=CHC = CHCH=CHC=CHCH = CHCH=CCH = CHCHr=CCH=CHCOOCH,

Bixin

Cyclization can take place at both ends or at one end of the chain. By
oxidative degradation the chain may be shortened. On the other hand, the
introduction of various oxygenated groups gives rise to a series of derivatives.
The carotenes (carrot, lemon, orange, apricot, etc.) and lycopene (tomato) are
hydrocarbons, whilst cryptoxanthin (fruit of Chinese lantern plant, egg yolk,
etc.), rubixanthin (berries of the wild rose, etc.), xanthophyll (green and
yellow leaves, dandelion flowers, eggyolk, etc.), lutein (pumpkin, many yellow
flowers, etc.), zeaxanthin (maize grain, egg yolk, etc.), flavoxanthin (buttercup
flowers, etc.), violaxanthin (laburnum flowers, etc.), taraxanthin (dandelion
flowers, etc.) and fucoxanthin (algae of the genera Fiicus, etc.) are alcohols.
Similarly, rhodoxanthin (berry of the yew-tree, etc.) is a diketone, capsanthin
(red pepper) and capsorubin (red pepper) are hydroxy-ketones and crocetin
(saffron or the pigment from Crocus pollen, etc.) is an acid. Carotenoid
pigments are widely distributed in the biosphere and will be found, no
matter whether one examines plants or animals, algae or bacteria.

REFERENCES

Deuel, H. J. (1951) Carotenoids and related compounds, in The Lipids, vol. I,
Interscience, New York.

Goodwin, T. W. (1952), The Comparative Biochemistry of the Carotinoids. Chap-
man and Hall, London.

Karrer, p. & JucKER, E. (1948) Carotinoide. Birkhauser, Basel.

38 UNITY AND DIVERSITY IN BIOCHEMISTRY

B. Natural Phenanthrene Derivatives

There are a number of substances occurring naturally that, from the
organic chemists' point of view, are related to phenanthrene. This latter
substance, Cx4Hio, is obtained from coal tar together with anthracene, of
which it is an isomer.

Among the phenanthrene derivatives present in the biosphere, one finds
many quininoid pigments and a number of alkaloids such as those of the
morphine group. But the phenanthrene derivatives most characteristic of
living organisms are those in which the phenanthrene nucleus has a cyclo-
pentane ring fused to it.

CH
HC CH

CH C CH

// \ / \ /
HC C C

HC C CH

\ / \ /
CH CH

Phenanthrene

If a hydrogen atom is added to each carbon of the phenanthrene ring the
double bonds disappear and one obtains perhydrophenanthrene.

H,

c

/ \

H2C CH2

H,

C H CH CH2

/ \ / \ /

H2C C C H

HoC C CH2

\ /

\ /

c

C

H2 H H2

Perhydro

phenanthrene

H2

c

/ \

H2C CH2

I I

Cyclopentane

Both the structures of cyclopentane and of perhydrophenanthrene are
combined in cyclopentanoperhydrophenanthrene, commonly known as
sterane.

CONSTITUENTS OF THE BIOSPHERE

39

C

CH,

/"W"\

H:.C,, ijC

H
H, \

C' H C

9

c

16

CH,

D

H,C, "C\„ ^CH

14 C-

15

CH,

H.C

B

sC

CH,

AA

C H C
Ho Hj

The natural compounds containing this ring, the steroids, are oxy-
genated, alkyl-substituted derivatives of the hydrocarbon, having the
following general formula, where R and R' vary from compound to com-
pound :

derived from oestrane

CH,

In the biosphere this basic carbon skeleton appears in a series of guises,
the most important being the following :

Substituent group R and R'
Cholesterol*

CHs

I
CH3-CH-(CH2)3— CH(CH3)2

* The other sterols have double bonds in the side chain or alkyl substituents.

40

Bile acids

UNITY AND DIVERSITY IN BIOCHEMISTRY

CH3 CH3

I

— CH— (CH2) 2— COOH

Androgenic substances CH3 OH or O

Luteal substances CH3 Acetyl

Oestrogenic substances (benzene ring) OH or O

1. Sterols

In the biosphere, sterols generally have a single hydroxyl group together
with a side chain.

Their general structure

CH3

HO

causes them to be classified, from the chemical point of view, as derived
from cholestane C27H48 or from coprostane (allocholestane).

CH

21CH3 26CH3

I ^

CH— CH2— CH2— CH2— CH

17\ 20 22 23 24 2S\

Cholestane

The molecule of cholestane contains eight asymmetric carbon atoms
(5, 8, 9, 10, 13, 14, 17, 20) : there are theoretically 256 stereoisomers. If a
hydroxyl group is attached to C-3, the number of stereoisomers is raised to
512. Most of the natural isomers differ only in the arrangement of groups
in the region of C-3 or C-5. In general, the methyl group on C-10 is
situated above the plane of the paper. If, as is generally the case, the
hydroxyl group on C-3 is also above the plane of the paper, the isomer is
designated cis or ^. In the inverse case, the compound is called trans or a.
When a single bond in cholestane is replaced by a double bond, it

CONSTITUENTS OF THE BIOSPHERE

41

becomes a cholestene and the position of the double bond is indicated by
the first (in the order of increasing numbers) carbon atom Hnked by the
double bond.

One of the most widely distributed sterols, for example, is cholesterol or
3 (m)-hydroxy-5-cholestene.

HO

CH2— CH2— CVU— CH

CH.

CH3

Cholesterol

The numerous sterols in the biosphere differ in the number and position
of the double bonds, and in the number of carbon atoms in the side-chain.
There are saturated sterols, and sterols having one, two or three double
bonds. In the higher animals, cholesterol is the characteristic sterol, but
this is not the case for lower animals, in which cholesterol is only one
sterol among many.

Ergosterol is the chief sterol in mushrooms, and fucosterol in the brown
algae. The higher plants contain complex mixtures of sterols. Bacteria,
in general, do not contain sterols, or other steroids.

Werner Bergmann classes the sterols according to their rotatory power,
which is intimately related to their structure :

system of conjugated double bonds in
ring B.

a 5:6 double bond.

I. [a] greater than —90'

II. [a] from -30° to -70°

III. [a] from -20° to +10°

IV. [a] from +10° to +30°

V. [a] from +40° to +50°

a 7:8 double bond

saturated ring and side chain (excep-
tion : neospongosterol).

an 8:9 double bond.

The sterols in group I differ from all the others in one important respect :
the conjugated double bonds in ring B confers upon them the property of
undergoing molecular rearrangement under the influence of ultra-violet
light.

Ergosterol, the chief sterol of mushrooms, belongs to this group, the
members of which differ from each other in the side-chain (presence or

42

UNITY AND DIVERSITY IN BIOCHEMISTRY

absence of double bonds, position of methyl and ethyl groups, etc.). Simi-
larly, it is the character of the side-chain which differentiates the sterols of
group II (cholesterol, camposterol, clionasterol, etc.).

2. Bile Acids

These substances, so named because they were isolated from the bile of
vertebrate animals, are cousins to the sterols of group IV and they possess
two methyl groups and at least one hydroxyl in the ring system. Their C-5
side-chain ends in a carboxyl group. Cholic acid is an example :

CH:,

CH, — COOH

OH

CH3 c— CH,

HO

Cholic acid

3. Other Natural Steroids

There exist in the biosphere steroids having a chain of only two carbon
atoms attached at position 17. These are the 21 -carbon steroids. They are
derivatives of pregnane. Progesterone and the mammalian adrenocorti-
costeroids fall into this category.

Other steroids do not have a side-chain attached to carbon 17. They
usually carry two methyl groups and are C-19 steroids derived from aetio-
cholane. Among them are testosterone and those steroids which have a
keto group in position 17 and are consequently called 17-ketosteroids; they
are often present in mammalian urines.

CH,

Aetiocholane

CONSTITUENTS OF THE BIOSPHERE

43

Other steroids not possessing a methyl group arc the C-18 steroids,
derivatives of oestrane. The most interesting of these are the oestrogenic
hormones in which ring A is a benzene ring (e.g. ©estrone).

O

CH3 CHJI

HO

Oestrane

Oestfone

C. Natural Heterocyclic Compounds
(a) Pyrrole Derivatives

Among the heterocycHc compounds of organic chemistry, those of the
pyrrole group present a particular interest to the bicohemist. Pyrrole itself,
C4H4NH, has been known for over a century as a product of the dry
distillation of organic matter. Its biological importance is very great.
Suffice it to say that from indole or phenopyrrole are derived indoxyl and

H H H

H— C C-H

il l|

H — C C-H

\ /
N
H

Pyrrole

REFERENCES

Bergmann, W (1952). Sterols. Progress in the Chemistry of Fats and Other Liquids,

1 , Pergamon Press, London & New York.
FiESER, L. & FiESER, M. (1949). Natural Products Related to Phenanthrene, 3rd

ed., Reinhold (New York).
Heusghem, C. (1950). Metabolisme et analyse des hormones steroides, Masson and

Desoer, Paris, Liege {Actualites biochimiques. No. 14).

44

UNITY AND DIVERSITY IN BIOCHEMISTRY

indigo, that considerable amounts of pyrrole bases are present in plants,
and that tryptophane contains a pyrrole ring. One particular class of pyr-
role derivatives, the porphyrins, are of great interest due to their wide
distribution both in the free and in the combined state. The porphyrins
crystallize readily, have a very high melting point and fluoresce brilliantly
in ultra-violet light. Their colouring power is extremely great and spectro-
scopic analysis allows very small quantities of porphyrins to be detected.
They are molecules containing four pyrrole rings and their structure may
be represented as based upon a hypothetical tetrapyrrole, porphin, the
structure of which in each particular case is modified by the addition of
various groups. The position of the double bonds can only be arbitrarily
fixed in a structure of this sort.

The four pyrrole rings of porphin and the porphyrins are connected by
methyne bridges (= CH — ) which are denoted by the Greek letters a, j8,
y and 8. The porphyrins are derived from the parent porphin nucleus by
replacing the hydrogen atoms on carbons 1 to 8 by various radicals.

The relative positions of the substituent groups can give rise to several
isomers. Let us consider, for instance, in one category of porphyrins, the
tetramethyl-tetrapropionyl-porphins, also called coproporphyrins. There
are four possible isomers corresponding to different positions of the four
methyl groups :

1 : 1, 3, 5, 7-

II : 1, 4, 5, 8-

III : 1, 3, 5, 8-

IV: 1, 4, 6, 7-

These four isomers have the following structures :

HOOC - H.C - HX H CH

H,C

HOOC - CH, - HjC

CH, - CH, - COOH

CH, H CHj - CHj - COOH

CONSTITUENTS OF THE BIOSPHERE

45

HOOC - H,C - H,C H CHj - CH^ - COOH
H,C<^ r if ^CH,

HOOC - H,C - H,C

CHj - CHj, - COOH

HOOC - H,C - H,C

HOOC - H„C - H,C

HOOC - H,C - H,C

H CHj - CH, - COOH

^ CHj - CH, - COOH

CH,

H

CHj- CHj -COOH

CH

CH,

H ^"3

Coproporphyrin I is found in yeast, and coproporphyrins I and III have
been isolated from faecal matter, from which they take their name.

CHj

HC 11 ^u. H H CH,

CH,

CH=CH,

CH,

CHj CH,

I I

COOH COOH

Protoporphyrin

CH,

I

COOH

Deuteroporphyrin III

46 UNITY AND DIVERSITY IN BIOCHEMISTRY

In the protoporphyrins, the eight carbon atoms of porphin carry two
vinyl groups, four methyl groups and two propionic acid groups. One of
the fifteen possible isomers of this structure is protoporphyrin IX or
porphin-l:3:5:8-tetramethyl-2:4-divinyl-6:7-dipropionic acid, commonly
called protoporphyrin. This is the porphyrin most widely distributed in
nature, it is a constituent of haemoglobin, catalase, peroxidase, of cyto-
chrome-6, etc. During the course of putrefaction protoporphyrin loses its
unsaturated side chains and is transformed into deuteroporphyrin III.
On the other hand, it can be derived by a series of operations from copro-
porphyrin III, another proof of its chemical kinship with the porphyrins
of Series III.

[b) Derivatives of Pyrrolidine

Pyrrolidine is the saturated ring compound corresponding to pyrrole.

H2C CH2

\ /
H

N

Pyrrolidine

Its skeleton is found in molecules present everywhere in the biosphere,
as in proline, vitamin Bjg, nicotine and in other alkaloids, etc.

(c) Indole Derivatives

Indole is the compound whose ring is present in tryptophan. It is also
present in the natural auxin a-indolyl-acetic acid, a substance produced at
the level of the apical bud and of the young leaves in plants, and transported
to the region of the stem when growth takes place.

H

HC C CH ^^\

HC C CH ^^ C C-CH.COOH

H H C N^

Indole H H

a-indoyl-acetic acid

(d) Imidazole Derivatives

The five-membered imidazole ring containing two nitrogen atoms is
present in the naturally occurring amino acid, histidine.

CONSTITUENTS OF THE BIOSPHERE 47

N CH N CH

!l II 11 il

HC CH HC CH

\ / \ /

N S

H

Imidazole Thiazole

(e) Derivatives of Thiazole

This five-membered ring, containing both nitrogen and sulphur, to-
gether with a pyrimidine ring, forms part of the structure of thiamine
pyrophosphate which is an important coenzyme.

(/) Thioctic Acid

Another important sulphur-containing heterocyclic ring is that of
6:8-dithio-«-octanoic acid otherwise known as thioctic or a-lipoic acid.

S — CH2
CH2

I Thioctic acid

S— CH

I

(CH2)4

COOH

This substance, an important coenzyme for oxidative decarboxylation,
operates by opening and closing of its ring at the two sulphur atoms.

I -) i_r

,/^^l (CH.O.-COOH -* /\ (CH.0,-COOH

s-s ^ZYu ^^ ^^

Oxidized thioctic acid Reduced thioctic acid

{g) Derivatives of Six-membered Heterocylic Rings containing Oxygen

The principal rings in this category are those of pyran, chroman, and
flavan. The pyran ring, as has already been stated (p. 19) is the basic
skeleton of the sugars in their stable forms.

48

UNITY AND DIVERSITY IN BIOCHEMISTRY

C

/ \
HC CH

II II

HC CH

\ /
O

Pyran

H H2

Cv .c

^ \ / \

HC C CH

I II II

HC C CH

% / \ /
C O

H

Chroman

H H2

^ \ / \
HC C CH

H

H

HC C C-C^

CO \.

H "

Flavan

=c

H

CH

Chroman derivatives, called tocopherols, are widely distributed in the
biosphere. Although their metabolic role is not yet precisely defined, it is
known that they antagonize certain oxidations. The vitamins E are toco-
pherols.

CH,

CH,
I
- CH, - CH, - CHj -CH - CHj - CH, - CH, - CH'

C - CHj - CHj - CHj - CH - <_i-ij - v-n, - ^n, -».. . - v. .j
o . Tocopherol

CH,

CH,,

CH,

HO

H

H,

C
\

CH,

CH,

CH,

o/|

,C - CH, - CH, - CH, - CH - CHj - CH, - CH, - CH - CHj - CH, - CH^ - CH

CH,

CH-

CH,

CH,

B • Tocopherol

In plants there is a family of yellow pigments, the anthoxanthins, which are
derived from the flavan ring, from flavone, flavonol and flavanone.

CONSTITUENTS OF THE BIOSPHERE

49

H O

^\ /^\ H H

HC C CH C-C,

I 1 II 2 II ^ ^

HC C C— C 3 CH

% ^ \ X \ /^

C O C = C

H H H

Flavone

H O

-iJj^ \ / \

HC C CH.

I 1 I! 2 I

HC , ,C C — C

H

Flavanone

H
C

H
C

3 CH

C= c-^

H H

H O
C C

H H
HC" 'C ^C-OH C — C,

HC^ C '^ C — C 3 CH

c o c=c

H H H

Fl

avonol

These compounds are generally found in nature in the hydroxyl substituted
form, to give such derivatives as quercetin, a flavonol derivative which in
combination with glucose (as a glucoside) is to be found in many plants, or
hesperitin, a flavanone derivative which is present in the lemon as a
glucoside, hesperidin.

OH o

hoAA

.OH

4^ OH

HO

Quercetin

OH

\

CH,

C

JDH

4>OCH3

Hesperidin

50 UNITY AND DIVERSITY IN BIOCHEMISTRY

Substances of this nature have a function in the transfer of metabolic
hydrogen in plants. In the process, hydrogen peroxide formed by the
combination of hydrogen and oxygen oxidizes the flavone derivative in the
presence of peroxidase, and in turn the oxidized flavone oxidizes a molecule
of ascorbic acid.

This function is only possessed by derivatives having two hydroxyls in
positions 3 and 4 in ring 3, as is the case for quercetin and its glycoside
quercitrine, in which the glucose molecule is combined with the hydroxyl
group of ring 1. Hesperidin is inactive.

However, in the lemon when it is ripe, a demethylation of hesperidin
takes place and eriodictyol is formed and it is the glucoside of this latter
substance which is chiefly found in the ripe fruit.

OH O
/V^CH, OH

"'^VAo-""^^^"

Eriodictyol

The anthocyanins, the blue, red or violet pigments found in many
flowers, are glucosides in which there is a six-membered oxygen-containing
ring. Their aglucones (non-sugar portion) or anthocyanidins are hydroxy-
lated derivatives of a benzopyrilium nucleus :

H H

C C ,

HC C CH

I II I

HC C CH

% y ^ <^

C 0_j-

H
in which oxygen is the central atom of a complex monovalent ion whose
coordination number is three. Certain anthocyanidins have a structure
similar to that of flavone, for example, this is the case with oenin, the
anthocyanidin present in the skin of the black grape.

H

H
HO — C. C^

I II I ^^ ^ — O— CH3

HO— C C C C^ ^C — OH

% ^ ^ ^ C=C— OH

C 0+ ^ '^ UH

H CI-

Oenin chloride

CONSTITUENTS OF THE BIOSPHERE 51

(h) Pyridine Derivatives

The simplest six-membered heterocyclic ring containing nitrogen is that
of pyridine. It can be prepared by cyclization of ethylallylamine followed
by dehydrogenation.

Its formula is :

H CH,=CH— CH,— NH— CH,— CH3.

C \ ethylallylamine

HC5 3CH

II I

HC 6 2 CH

\ ^ ^

N

pyridine

It is present in the distillation product of bones and coal. It behaves
similarly to the benzene ring with regard to halogenation, nitration and
sulphonation, but the reactions are considerably slower. It is the ring
present in an important group of alkaloids. This group includes nicotine,
present in tobacco leaf in combination with malic and citric acids, and
which on oxidation yields nicotinic acid.

H

C^ HiC CHo

y % II

HC C HC CH2

II I \ /

HC CH N— CH3

N

Nicotine (Methylpyrrolidine-pyridine)

H H

y\

c

HC C — COOH y^(^ C— CONH2

» ' li I

HC CH HC CH

Nicotinic acid Amide of nicotinic acid (Nicotinamide)

■n''^ ^n^

The amide of nicotinic acid, or nicotinamide, is a substance of very great
biological importance, for it is the active grouping in a whole series of
important coenzymes. Another important group of pyridine derivatives
comprises pyridoxin (also known as vitamin Bg, being a growth factor for

52

UNITY AND DIVERSITY IN BIOCHEMISTRY

the rat and for certain bacteria), pyridoxal and pyridoxamine. These
substances are very widely distributed in nature.

HOH,C

CHoOH

OH HOH.C

\ /^CH3

CHO

OH HOHjC

Pyridoxin (vitamin Bg)

Pyridoxal

CHjNHa

OH

Pyridoxamine

{i) Pyrimidine Derivatives
In pyrimidine there is a heterocycHc ring containing two nitrogen atoms.
It is one of three isomeric diamines :

HC N

I I!

HC CH

^ /
C

H

Pyridazine

HC 6 2 CH

HC 5

3 N

C

H

Pyrimidine

^ \

HC CH

I II

HC CH

"^ /

N

Pyrazine

To the organic chemist, pyrimidine derivatives are products of the
condensation of urea with certain acids. Barbituric acid or cyclomalonyl-
urea is a member of this group. It is prepared by reacting malonic acid
with urea in the presence of POCI3.

COOH NH— CO

o = c<

Urea

NH.
NH,

+

CHo = OC

CHj + 2H2O

COOH

Malonic acid

NH— CO

Barbituric acid

1. Pyrimidines

Among the oxypyrimidines (their formulae, like those of the oxypurines,
may be also written as if they are hydroxyderivatives) are a number of
important constituents of nucleic acids, cytosine, uracil and thymine.

N = CH HN-C=0

I

0=C C— CH3 0=C C— H

1

HC 2

3

N

6 I
5 CH

4

HN— C=0

N=C— NH2

0=C CH

CH

Pyrimidine

HN— CH

Uracil
(2, 6-dioxy-
pyrimidine)

HN— CH

Thymine

(2, 6-dioxy-5-methyl-

pyrimidine)

HN— CH

Cytosine

(2-oxy-6-amino-

pyrimidine)

CONSTITUENTS OF THE BIOSPHERE

S3

2. Thiamine

This is the hydrochloride of a molecule formed from a pyrimidine and a
thiazole ring. The phosphoric ester of this substance is an important
coenzyme.

CH3

CH,

N=C— NH,
-C C CH.

N— CH

C=C— CH2CH2OH

\

S

/

N=C

+ H

CI-

Thiamine hydrochloride

(j) Purine Derivatives
The purine ring results from the condensation of a pyrimidine ring with
another heterocyclic ring, imidazole.

N =CH

, 1 s,

11 3 J'
N CH

HC — NH\

HC — N ^

Imidazole

CH

N=CH
I'M H
HC: sC — N\

N — G — N '^

Purine

Pyrimidine

Purine forms salts with acids and compounds with bases, it behaves there-
fore both as an acid and as a base.

A number of purines are widely utilized in living organisms.

/

NH,

O

//

N=:C

HC C-

H

-N

HN— C

I I H
NH,— C C— N

//

CH

N— C— N

Adenine
(6-aminopurine)

o

//

HN— C

I I H
HC C— N

^ CH

II II //

N— C— N //

Hypoxanthine
(6-oxypurine)

N— C— N

Guanine
(2-amino-6-oxypurine)

o

//

HN— C

I I H
0=C C— N

CH

HN— C— N

Xanthine
(2,6 dioxypurine)

\

CH

54 UNITY AND DIVERSITY IN BIOCHEMISTRY

(k) Alloxazine Derivatives
The double heterocyclic ring of lumazine,

/N. NH

HC ' C 2 CO

HC « C 3 NH

^ N ^ ^ CO
condensed with a benzene ring forms the alloxazine nucleus

H H

HC7 C C 2 CO

HC6 /^ rl^ '

HC 6 c C 3 NH

CH

N

this has been known for over half a century and the method of synthesis by
condensation of an aromatic o-diamine with alloxan (Kuhling's sythesis)
has also been known for a considerable time.

NH

H

N

/ \

' OC CO

-t- I I

alloxazine + 2 H2O

NHj OC NH

\ /

C

o

It was the great achievement of Richard Kuhn and his fellow workers
and then of Karrer and co-workers to show that a very important series of
natural substances contained a tautomeric form of alloxazine, isollaoxazine
or flavin, a substance which is unstable in the free state.

~ H H

CNN

HC7 C C 2 CO

I 11 I I

HC 6 C C 3 NH

C N C

H O

Isoalloxazine or Flavin (unstable)

CONSTITUENTS OF THE BIOSPHERE

55

Although unstable in the free state, the ring is present as derivatives in
which the hydrogen on the nitrogen atom in position 9 is replaced by a
substituent group. The group of these compounds is called "the flavins".
One example is lumiflavin (a photoderivate of lactoflavin).

H CH3

C N N

^ "- / \ ^ "^_
CH.C C C CO

CH3C C C NH

C N^ C

H O

6,7-dimethyl-9-methyl-isoalIoxazine or lumiflavin

(/) Pteridine Derivatives

This heterocyclic ring system arises from the condensation of the pyrimi-
dine and the pyrazine rings.

6
Pteridine

The pteridines are sparingly soluble in water and insoluble in volatile
organic solvents. They have a bright fluorescence.

1. Pteri?is

The pteridine nucleus is present everywhere in the biosphere, in the
form of a number of derivatives : leucopterin, xanthopterin, erythropterin,
etc. Pterins are found, especially, in the wings of butterflies. The fluores-
cence of tissues is often due to their presence.

2. Folic Acid

Also among these derivatives is pteroyl - glutamic acid or folic acid, so
called because of its abundant distribution in the leaves of plants. Folic acid
is the universal coenzyme for the transfer of C^ fragments and hence is
indispensable for the synthesis of purines and nucleic acids.

56 UNITY AND DIVERSITY IN BIOCHEMISTRY

2-amino-4-hydroxy para-aminobenzoic glutamic acid

-6-methylpterin acid

OH

I
C N

/ \ / \

O H H

N C C-CH,-N<' ^-C-N-C-CH,-CH,-COOH

I I COOH

H,N-C C CH

y

pteroic acid

Y

pteroylglutamic acid (folic acid)

REFERENCES

Bendich, a. (1955). Chemistry of purines and pyrimidines, in E. Chargaff et

J. N. Davidson The Nucleic Acids, Vol. I, Academic Press, New York.
Lemberg, R. (1954). Porphyrins in nature, Fortschr. Chem. org. Naturstqffe, XI,

299-349.
Sebrell, W. H. et Harris, R. S. (1954). The Vitamins, Vol. Ill, Academic Press,

New York.
Wiley, R. H. (1953). Heterocyclic chemistry, in H. Oilman, Organic Chemistry:

An Advanced Treatise, Vol. 4, Wiley, New York.

CHAPTER III

MODES OF LINKAGE BY COVALENT BONDS

I. "OSIDE" LINKAGE

A. OSIDES

On hydrolysis, osides yield one or more sugars or "oses". They are called
holosides if the products of hydrolysis are solely sugars, and heterosides if
on hydrolysis substances other than sugars are obtained.

The holosides are designated di-, tri- or tetraholosides according to the
number of sugar molecules obtained on hydrolysis.

The heterosides are very abundant in the vegetable kingdom: tannins, rube-
rythric acid (from the madder plant, hydrolysis Hberates glucose and the
aglucone, alizarin), anthocyanins (colours of many flowers), digitalis gluco-
sides (gitine, digitonine, digitaline, gitoxine, etc. . . .) cyanogenetic glucosides
(whose hydrolysis liberates hydrogen cyanide in addition to a sugar), etc. . . .

The heterosides are called a or jS-heterosides according to whether they
contain the a or j8 form of the sugar.

The most interesting osides in the biosphere are the diholosides or disaccha-
rides which are classed as reducing or as non-reducing disaccharides.

(a) Non-Rediichig Diholosides

CHjOH

a-Glucopyranose

HOH.C

CH.OH
Q^ /3-Fructofuranose

[a]^ - + 66,67°
sucrose

57

58

UNITY AND DIVERSITY IN BIOCHEMISTRY

1. Sucrose

This is the sugar of the sugar-cane and sugar-beet. It is present in the
tissues and juices of many plants (carrot, beetroot, sweet fruits, sugar-
maple juice, sugar-cane, etc. . . .)

In the molecule of sucrose, glucose is present in the a-glucopyranose form
and fructose in the /3-fructofuranose form. The two sugars are joined by their
two reducing groups and as a result sucrose has no reducing properties.

(b) Reducing Diholosides
1. Lactose

CH.OH

CH,OH

OH

H OH

jS-Galactopyranose

H OH

;8-Glucopyranose

Lactose (/3 form)

This is milk-sugar, it is present in the milk of all mammals (4% in cows
milk, 5-7% in human milk).

Lactose is dextrorotatory, it exists in an a form and in a ^ form according
to the configuration of the remaining free pseudoaldehyde group.

2. Maltose

CH.OH CH,.OH

o.

HO

H

H

HO

OH

H HO

a-Glucopyranose

H

HO

/3-Glucopyranose

J

Maltose (/3 form)
It is obtained when amylase acts on starch or glycogen ; it is dextrorotatory.

3. Cellohiose "

CHoOH

H OH

j3-Glucopyranose

H OH

|3- Glucopyranose

Cellobiose (j8 form)

MODES OF LINKAGE BY COVALENT BONDS

59

It is a product of the hydrolysis of cellulose, like lactose and maltose, it
exists in an a and ^ form.

B. Nucleosides

These substances are the result of the combination of a base (most
frequently a purine or pyrimidine base) with a sugar (D-ribose) or desoxy-
sugar (desoxyribose) by means of an "oside" bond. Since many of these
substances occur in nucleic acids, they are called "nucleosides".

In these compounds the linkage is probably a /S-glucoside type of linkage.

N=C— NH2

HC C— N

N— C— N

CH

CH

H— C— Ori

H— C— OH

O

H— C

CH2OH

Adenosine (adenine nucleoside)

HN— CO

HoN— C C— N

\

CH

N— C— N ^ CH

H-C-OH

O

H— C— OH

H— C

CH2OH

Guanosine (guanine nucleoside)

Among the flavins, or isoalloxazine derivatives, one finds an important
natural derivative of this type, it is lactoflavin — more commonly known as
riboflavin. Chemically, it is 6,7-dimethyl-9-D-ribityl-isoalloxazine. It is
present in a combined state in a large number of animal and vegetable
tissues. Like the flavins in general, riboflavin is soluble in water, giving a

60 UNITY AND DIVERSITY IN BIOCHEMISTRY

yellow solution having a yellow-green fluorescence. With heavy metals it
forms sparingly soluble salts. When heated, it decomposes at 274°. It is
stable to oxidizing agents. Under the influence of light, it is transformed,
depending on the conditions, either into lumilactoflavin or lumiflavin (a
derivative of isolloxazine, p. 54), or into lumichrome (a derivative of
alloxazine). Riboflavin, or vitamin B^^, is not a true nucleoside, since the
isoalloxazine in it is not combined with ribose, but with ribitol, the
corresponding alcohol.

H H

I \

CNN

<^ \ / ^ / \

H..C— C C C CO

I II i I

H.C— C C C NH

% y ^ ^ \ y

C N C

I II

H O

Lumichrome

H

HO— C— H {">')
I
H— C— OH (4')

H— C-OH (3')

H— C— OH (2')

I
H— C— H (!')

H

CNN

HaC— C 7 C C 2 CO

^ I II I I

H3C— C 6 C C 3 NH

6,7-dimethyl-9-D-ribityl-isoalIoxazine or vitamin B2

REFERENCES

Baddiley, J. (1955). Chemistry of nucleosides and nucleotides, in E. Chargaff
and J. N. Davidson, The Nucleic Acids, Vol. I, pp 137-190 Academic Press,
New York.

PiGMAN, W. W. & GoEPP, R. M. (1948) Chemistry of the Carbohydrates, Academic
Press, New York.

MODES OF LINKAGE BY COVALENT BONDS 61

II. ESTER LINKAGE (AND ANHYDRIDE LINKAGE)

A. Ternary Lipides

Lipides are the esters which constitute fats. A distinction is made be-
tween ternary Hpides, containing only carbon, hydrogen and oxygen, and
complex lipides containing in addition phosphorous and nitrogen. These
latter compounds are better considered with the other natural phosphate
esters.

The ternary lipides can be divided into several types, among which are :

(a) The glycerides, esters of glycerol.

(b) The waxes, ester of higher alcohols.

(c) The sterides, esters of sterols.

(a) Glycerides

The simple glycerides are those in which the three molecules of acid,
which take the place of the H atom in each of the OH groups of glycerol,
are identical. The general formula of the simple glycerides is therefore as
follows :

CH2— O— OC— R

1
CH — O— OC— R

I
CH2— O— OC— R

In certain other glycerides, the mixed glycerides, the three molecules of
fatty acid are not identical, for example in distearopalmitin :

CHo- O— OC— CnHa,

i
CH — O— OC— CoH,,

CH,— O— OC— CH,-.

Distearopalmitin

(1 molecule of glycerol + 2 molecules of stearic acid + 1 molecule of
palmitic acid)

{b) Waxes

These are esters of the higher molecular weight fatty acids and mono-
valent higher alcohols.

Example : cetyl palmitate, the principal constituent of spermaceti
(cetyl alcohol = C16H34O).

62 UNITY AND DIVERSITY IN BIOCHEMISTRY

{c) Sterides

The sterides are esters of fatty acids and sterols. Lanoline, the fat ob-
tained from wool, is a mixture of cholesterol oleate, palmitate, and stearate.

B. Natural Phosphoric Esters

{a) Phosphoric Acids

Orthophosphoric acid, H3PO4, possesses three acid groups which ionize,
one after the other, as the pH increases.

OH

/

0=P— OH

\
OH

The first ionization corresponds to a pK of 1-97, so that this group is
relatively strongly acidic, and the ionization is complete at a very acid
pH, well outside the pH range of biochemical interest. The second acid
group (pK = 6-82) is comparable to organic acids in strength. It is this
group, when combined with a strong base, which acts as a buffer in the
acid-base equilibrium of biological environments. The third acid group is
only slightly dissociated, only forming salts in very alkaline solution, out-
side the biochemical range. Orthophosphoric acid can form phosphoric
esters with alcohols. Three types exist : monoesters, diesters and triesters —

OR OR OR

/ / /

0=P— OH 0=P— OR' 0=P— OR'

\ \ \

OH OH OR"

In the biosphere, triesters of orthophosphoric acid are unknown,
however, this does not exclude the possibility that this binding may be
present in certain macromolecules. The diesters of orthophosphoric acid
which exist in the biosphere are often mixed esters. Acid or alkaline
hydrolysis slowly transforms them into monoesters. Most of the complex
lipides are diesters and vitamin B^g also falls into this category. The mono-
phosphoric esters of alcohols form a very important biochemical group.
The two free acid groups are more strongly acidic than when they were

REFERENCES

Deuel, H. J. Jr. (1951) The Lipids. Their Chemistry and Biochemistry, Vol. I :

Chemistry. Inter-science, New York.
HiLDiTCH, T. P. (1947). The Chemical Constitution of Natural Fats, 2nd ed.,

Chapman and Hall, London.
LovERN, J. A. (1955). The Chemistry of Lipids of Biochemical Significance. Methuen,

London.

MODES OF LINKAGE BY COVALENT BONDS 63

present in orthophosphoric acid alone, so that these substances are stronger
acids than phosphoric acid itself. When phosphoric acid is liberated from
these esters and regains its three acid groups there is no appreciable change
in the reaction of the medium. Among the monoesters of orthophosphoric
acid, we might mention glucose-6-phosphate, a-glycerophosphoric acid
and fructose-6-phosphate.

Orthophosphoric acid may be esterified, not only with alcohol groups,
but also with the pseudoaldehyde groups of sugars. Aldose derivatives in
which the reducing group of the sugar is combined with orthophosphoric
acid are very important in cell-chemistry. Sugar- 1 -phosphates are in this
category ; their general formula is as follows :

PO3H2— O— C— (CHOH) , — CH— Pv
/

H

-0-

R=H

or CH2OH

They are very easily hydrolysed in strongly acid solution. The sugar- 1-
phosphates of furanose sugars are more acid-labile than those of the
pyranose form.

Phosphoric acid also forms esters with enols, the most interesting of these
is phosphopyruvic acid, in which the enolized pyruvic acid is combined
with H3PO4. Very acid-labile and very alkaline-labile, it is readily split by
oxidizing agents liberating phosphoric acid.

Phosphoamides are also phosphoric acid esters. Their general formula is :

HO R

\ /

P— N

/w \

HO O R'

Phosphoamides of the phosphoguanidine type are very acid- and alka-
line-labile and their rate of acid hydrolysis is increased by molybdic acid.
Phosphoarginine and phosphocreatine are phosphoamides.

Orthophosphoric acid can associate with other molecules of the same acid
by means of anhydride linkages to form polyphosphoric acids, notably
pyrophosphoric and triphosphoric acids.

Pyrophosphoric acid is made up of two molecules of ortho-phosphoric acid.

HO OH

\ /

P— O— P

/W !l\

HO O O OH

64 UNITY AND DIVERSITY IN BIOCHEMISTRY

It is very stable in alkaline solution, but not in acid solution. A large
number of important biochemical structures are derived from pyrophos-
phate, similarly triphosphoric acid also plays an important role in bio-
chemical energetics in the form of its derivatives, adenosinetriphosphate
(ATP) and uridinetriphosphate (UTP).

(b) Phosphoric Esters of Glycerol and Glyceric Acid

The monophosphoric ester of glycerol, or glycero-phosphoric acid,
exists in the two isomeric forms a and ^ :

CHoOH CHoOH

I I

CHOH CH— O— PO3H2

I I

CH2— O— PO3H2 CHoOH

P

Glycerophosphoric acids

and corresponding to these are the monophosphoric esters of glyceric acid,
a and ^ phosphoglyceric acids,

CO2H CO2H

1 1

CHOH CH-O-PO3H2

I I

CHo— O— PO3H2 CHoOH

3 or j8 2 or a

Closely related to the above acids is phosphorylated pyruvic acid
(phosphoenolpyruvic acid) :

CO2H

I
C— O— PO3H2

II
CH2

(c) Triosephosphates

Phosphodihydroxyacetone CH2OH-CO-CH2O-PO3H2 is a triose ester
a triosephosphate. Another member of the same class is its isomeric
aldehyde, phosphoglyceraldehyde or 3-glyceraldehyde-phosphoric acid :

CHO— CHOH— CHo— O— P0(0H)2

These two triosephosphates, unlike the hexose phosphates, are easily
hydrolysed by alkali.

MODES OF LINKAGE BY COVALENT BONDS

65

(d) Phosphor ylated Sugars

1. Ribose phosphates

The ribose phosphate normally present in cells is ^-D-ribose-1 -phosphate;
it is very acid-labile. Desoxyribose-1 -phosphate is even more unstable in
acid solution; at pH 4-0 at room temperature, it is 50% hydrolysed in 15
minutes. It is hydrolysed in the course of estimations of "inorganic phos-
phate" and is consequently often measured as such.

\

O H

\ /

C

HC

HCOH O

HCOH

PO3H,

\
O

H

\ /
C —

CH2

HCOH

HC

O

CH.OH

^-D-ribose-l -phosphate

CH2OH
Desoxyribose- 1 -phosphate

2. Hexose phosphates

(a) oc-D-glucose-\-phosphate [Cori ester) — This ester is reducing and is
stable to alkali, on hydrolysis glucose is liberated.

CH.OH

O H

O - PO,H,

OH

Glucose-1 -phosphate
Cori ester

CH;,0 - POjH,

OH

Glucose-6-phosphate
Robison ester

HiOjP-OHjC

CHjOH

OH

Fructose-6-phosphate
Neuberg ester

HjOaP - OH;.C

CH.OP03H,

OH

Fructose-1 ,6-diphosphate
Harden and Young ester

F

66

UNITY AND DIVERSITY IN BIOCHEMISTRY

(b) Glucose-6-phosphate {Rohison ester) — Although the existence of this
ester had been known since 1914 from experiments on yeast carried out by
Harden and Robison, it was not until 1931 that Robison and King suc-
ceeded in obtaining it in the pure state.

{c) Glucose-l,6-diphosphate — This ester, which is a coenzyme in gly-
colysis, was isolated by Leloir from yeast after incubation with phosphate
and glucose.

(d) Fructose-1 -phosphate (Robison ester) — Hydrolysis of fructose- 1,
6-diphosphate by phosphatases, gives equal amounts of fructose- 1-
phosphate and fructose-6-phosphate. Fructose-1 -phosphate has been
isolated from liver and been found in the intestine during the intestinal
absorption of fructose.

(e) Fructose-6-phosphate (Neuberg ester) — Fructofuranose-6-phosphate
was first prepared by partial hydrolysis of fructose-1, 6-diphosphate and
later isolated from the products of alcoholic fermentation. In normal acid,
at 100°, the phosphate group in position 1 is split off about a dozen times
more rapidly than the group in position 6.

(/) Fructose-l, 6-diphosphate (Harden and Young ester) — Fructofura-
nose-1, 6-diphosphate in alkaline solution (0-2N NaOH) at 100° liberates
the whole of its phosphate in three minutes.

(g) Other phosphorylated sugars and their derivatives present in the
biosphere — Such compounds are galactose- 1 -phosphate, ribitol phosphate,
sedoheptulose phosphate, gluconic acid phosphoric ester, etc.

(e) Pyridoxal-5-Phosphate and Pyridoxamine-5- Phosphate

These are important coenzymes, the former in the decarboxylation of
amino acids, in transaminations, deamination of hydroxyaminoacids and in
the removal of sulphur from amino acids containing sulphur.

O

I!

HO-P-OH

6 c«o

H,C

OH

^CH3

Pyridoxal-5-phosphate

o

II

HO-P-OH

^ CH.NH,

H2C

OH

l.,^^"

Pyridoxamine-5-phosphate

MODES OF LENKAGE BY COVALENT BONDS 67

(/) Thiamine Pyrophosphate
[Cocarboxylase, Diphosphothiamine, DPT)

Thiamine, being basic, gives a series of salts and esters.

CHs O- OH

! I I

C=C— CHo— CHoO— P— O— P=0

\ II I

N=rC— NHo S O OH

I I /

CH3-C C CH N=C

nil + H

N-CH

The most important ester is the product of the reaction between pyro-
phosphoric acid and the hydroxyl of the thiazole ring. This ester is the
coenzyme for carboxylase and for the decarboxylation of a series of a-keto-
acids.

(g) Nucleotides
These are the phosphoric esters of nucleosides.

1. Mononucleotides

(a) Adenosine mo?io- and poly- phosphates — In most cells these com-
pounds act as coenzymes in the transport of phosphate groups.

Adenosine triphosphate (ATP) was isolated from muscle by Lohmann in
1928. The three terminal phosphate groups of ATP are joined by two
anhydride bonds. Removal of the terminal phosphate gives adenosine
diphosphate (ADP), and of the next phosphate leaves adenosine mono-
phosphate (AMP) or adenylic acid.

The adenosine phosphates (AMP, ADP, ATP) are relatively unstable
in solution. At 100° in dilute acid the two anhydride bonds of ATP are split
but the ester linkage remains intact. In AMP the esterification of the
adenosine is on C-5' of the sugar whilst in other nucleotides (coenzyme A,
triphosphopyridine-nucleotide) other carbon atoms are involved.

In the molecules of adenosine polyphosphates, one of the phosphoric
acid residues is linked to the nucleoside by an ester bond but the phos-
phoric acid residues among themselves are joined by an anhydride bond
much less stable than the ester bond. As we shall see the hydrolysis of
anhydride linkages plays an important part in biochemical energetics on
account of their strongly exergonic nature.

(b) Uridine phosphates— These mononucleotides have been demonstrated
in yeast and in animal liver. They are coenzymes for the reaction galac-
tose-1-P ±;: glucose- 1-P, and for the formation of sucrose from glucose and
fructose. As with the adenosine phosphates, a uridine-5 '-triphosphate

68

UNITY AND DIVERSITY IN BIOCHEMISTRY

(UTP), a uridine-5 '-diphosphate (UDP) and a uridine-5 '-monophosphate
(UMP), exist.

ATP

ADP

AMP

Adenosine

Adenine

N=C— NH2

HC C— N

\

CH

N— C— N

OH OH

C— C-

H H

-C— C— CH2— O-
H H

-P— O-

II
O

-P— O-

II

o

OH

I

-p=o

I

OH

HOC=N

I I
HC C=0

II I
HC— N-

OH OH

C— C C— C— CH2— O-

H H H H

Uracil

OH

I
-P— O-

II

o

OH

I
-P— o-

II

o

OH

I

-p=o

I

OH

Uridine

UMP

UDP

UTP

(c) Uridine diphosphate glucose (UDPG) — This nucleotide is the coen-
zyme of the isomerase which transforms galactose- 1-P into glucose- 1 -P.
It was discovered by Leloir, and has been isolated both from yeast and from
animal tissues so that it appears to be of general importance. A whole

MODES OF LINKAGE BY COVALENT BONDS

69

series of compounds exists, with structures similar to UDPG, in which the
glucose is replaced by other substances such as acetylglucosamine, for
example.

HOC=N

HC C=0

HC— N

CH.,OH

HC

HCOH

HC

HCOH

O

o

HCOH

HC

CH2

o —

HOCH

OH

I
-P-

O

OH

I

i

-P-

HCOH

I
HC

— O

o

UDPG

O

{d) Flavin monophosphate — This is commonly called flavin mononucleo-
tide (FMN) but this is incorrect since it is a compound of phosphoric acid
with riboflavin which is not a true nucleotide. The phosphate is attached
at the 5' position of the ribityl residue. Flavin mononucleotide is the
coenzyme of L-amino oxidase and of the TPNH-^Og transhydrogenase.

OH OH OH o

III I

CH2-C-C-C-CH2-O-P-OH

III I

H H H OH

H3C
H3C

v ^=o

^

N

NH

O

70 UNITY AND DIVERSITY IN BIOCHEMISTRY

(e) Nicotinamide mononucleotide —

CONH,

O

C- C - CH,- O- P- OH

OH

H H H H

This has been isolated from yeast.

2. Dimicleotides

{a) Diphosphopyridine-nucleotide (DPN) and triphosphopyridine-nucleo-
tide (TPN) — Diphosphopyridine nucleotide (DPN, coenzyme I, code-
hydrogenase I, cozymase), an important coenzyme of general utility,
contains the heterocyclic bases adenine and nicotinamide and two mole-
cules of D-ribose. The two component nucleotides are joined by a pyro-
phosphate bridge. A related compound, also widely distributed in the
biosphere, only differs from the above substance by having an additional
phosphate residue esterified at C-2' of the ribose molecule attached to
adenine. This substance is known as triphosphopyridine nucleotide
(TPN, coenzyme II, Co II or codehydrogenase II).

,f^

^

\

CONH,

HC

H,NC = N

I I
/N — C CH

+ N

I
HC

I
HCOH

I
HCOH

i
HC

I
HC-

•C-N

O

CH, OH OH

I I I

o — p — O— P —
II II
o o

DPN

HCOH

I
HCOH

HC —
I

CH,
I
— O

O

CONH,

H,NC - N

>N^

HC

I
HCOH

I O
HCOH

HC

I

CH,
1
O—

OH
I
-P —

II
O

O-

TPN

The pyridine nucleotides and their barium salts are very soluble in water.
The oxidized forms are written DPN+ or TPN+. DPN+ has two phosphate
residues bearing two primary acidic groups, whilst TPN+, has three
primary acid groups and, in addition, a secondary group.

Reversible reduction of the pyridine ring transforms DPN+ into the
reduced dinucleotide DPNH and, similarly, for TPN+.

MODES OF LINKAGE BY COVALENT BONDS

71

H

AH2 +

H

H

A

N
I
R

HH

CONH,

H

H

A +

H

\ /

N

I

R

CONH2

+ H

+

A stoichiometric transfer of hydrogen takes place, from the substrate to
the coenzyme, with the hberation of one equivalent of acid. In effect, a
highly basic quaternary nitrogen atom is transformed into a feebly basic
ternary nitrogen. The reduction takes place, as can be seen from the for-
mula, in the para position of the pyridine ring.

(6) Flavin adenine dinucleotide {FAD) — Like FMN, FAD is a pseudo-
nucleotide containing adenylic acid joined via a pyrophosphate linkage
to FMN.

H,NC = N

HC

,N-C CH

II U
N-C — N

OHOHOH

I I I

O

o

O-

OH

I

CH5-C-C-C-CH,-0-P-0-P-0-CH,-C— C-

I

I

OH OH

I I
H H

OH

I

-c-c

I I

H H

FAD

It is the coenzyme of xanthine oxidase, aldehyde oxidase and other aerobic
dehydrogenases. Like riboflavin and FMN, FAD is universally present
in the biosphere. It is reddish-yellow in colour but, like FMN and ribo-
flavin, its solutions are a yellow-green.

The formula above is that of the oxidized dinucleotide, reduction takes
place in the isoalloxazine ring as follows :

H3C

H,C

R

!

R

1

H

f^

f^

= 0

+ 2H

H5C

/\

/N

-0

V

^nA^^"

- 2H

"'^V^

II

0

H

0

72

UNITY AND DIVERSITY IN BIOCHEMISTRY

(h) Coenzyme A (Co A)

This essential compound is universally distributed. It is formed by the
joining of adenosine-3, 5-diphosphate, pantothenic acid-4'-phosphate and
thioethanolamine (cysteamine).

Cysteamine

KC

CHj-SH

Pantothenic

acid-4'-
phosphate

OH

Adenosine-3, 5-diphosphate

The hydrogen atom of the -SH group at the cysteamine end of coenzyme
A may be substituted by an acetyl group to give "active acetate" or acetyl-
Co A. The metabolic role of "active acetate" is a primary one, for it acts as
a universal donor of acetyl groups. It contains an acylmercaptan bond,
a carboxyl group and a sulphhydryl group being condensed together, with
loss of water, in an anhydride linkage. This is a so-called "energy-rich"
bond, its hydrolysis setting free about 16,000 calories per mole. Despite this
strongly exergonic hydrolysis, the acylmercaptan bond of C0A-S-CO-CH3
is very stable in aqueous solution at physiological pH's. It is only in the
presence of specific enzyme catalysts that the bond is hydrolysed.

(/) Cyanocohalamin {Vitamin B^^

Cyanocobalamin is widely distributed in living organisms ; it is found in
bacteria, in algae and in animal tissues, but it does not appear to be present
in the green leaves of plants. For man, it is an important vitamin, being
one of the "extrinsic factors" of haemopoiesis. It was crystallized in 1948;
the crystals are dark red, melt at 320° and their solution has well pronounced
absorption bands at 278, 361 and 550 m/x. It contains cobalt and phos-
phorous and the molecular weight is around 1,500. On acid hydrolysis,
cyanocobalamin yields 5,6-dimethylbenzimidazole, ribofuranose, phos-
phoric acid, l-amino-2-propanol and a cobalt complex in which the metal

MODES OF LINKAGE BY COVALENT BONDS

73

is surrounded by a hexacarboxylic acid, formed by the association of four
pyrrolidine rings modified by the inclusion of a conjugated system of
double bonds. The molecule contains six primary amide groups and one
secondary group joining the aminopropanol residue to the propionic acid
group of ring D.

Cyanocobalamin is a phosphoric diester in which the free acid function
is neutralized by the positive charge on the cobalt.

Cyanocobalamin, then, is the result of combination of adenosinemono-
phosphoric acid with a pyrrolidine chromogen via l-amino-2-propanol.

CH2CONH2

:H, H CH3 CH3 cHrCONH,

NH.CO-CH,
CH3
CH3

NH, CO-CH,

CO-CH2-CH

CH,-CH3-
CONH,

CH3 CHoCHoCONH^

HO-CHa

Cyanocobalamin (Formula of Todd et al.)

74 UNITY AND DIVERSITY IN BIOCHEMISTRY

(j) Complex Lipides

Most of the complex lipides are diesters of orthophosphoric acid. Those
not belonging to this category will be described with those that do. The
fact that the complex lipides described here are extremely widely distributed
in the biosphere, confers upon them the status of fundamental cellular
constituents.

1 . Glycerophosphatides

(a) Lecithins or phosphatidylcholines — Lecithins are esters of phos-
phorylcholine with glycerol which is esterified in the remaining two posi-
tions by fatty acids. Two isomers are possible according to whether the
binding is with the a carbon or the ^ carbon of the glycerol.

CH2— O— CO— Ri CH2— O— CO— R,

I I

CH — O-CO— R2 CH — O— P(0)— O— CH2-CH2— N(CH3)a

I
CHa— O— P(0) — O— CH2— CH2— N (CH3)3

I +

Q- CH2— O— CO— R2

I +

o-

a-lecithin j8-lecithin

Living matter only synthesizes a-lecithins which are to be found in all
cells. The existence of so many a-lecithins is due to the diversity of the
groups Rj and Rg which may be saturated or unsaturated. Choline, the
base present in lecithin, is also widely distributed in the biosphere :

(CH3)3N— CH2— CH2OH

The lecithins are insoluble in water, in which they swell up, but they are
soluble in alcohol and ether although insoluble in acetone.

(b) Cephalins or phosphatidylethanolamines — Cephalins are diacylgly-
cerylphosphorylethanolamines and they differ from the lecithins by the
substitution of choline by another base, aminoethanol.

NH2 • CH2— CHoOH

In the pure state, cephalins are soluble in methanol, ethanol, ether,
petroleum ether, chloroform, glacial acetic acid, and insoluble in acetone,
but when mixed with other glycerophosphatides (phosphatidylserine,
plasmalogens, etc.) they are insoluble in alcohol. It is likely that the
natural cephalins are of the a type, but this has not yet been completely
proved.

{c) Phosphatidylserine — These glycerophosphatides are soluble in chloro-
form but are less soluble in ethanol or methanol and this allows their

MODES OF LINKAGE BY COVALENT BONDS 75

isolation. Serine is the base which takes the place of choline, and the
phosphatidylserines have the following structure :

O

li
O CH-— O— C— R

II I

R— C— O— CH O

I II

CH2— O— P (O)— O— CH2— CH— COH

OH NH2

L — phosphatidylserine

(d) Plasmalogejis or acetalphosphatides — In the pure state, the acetal-
phosphatides are soluble in alcohol, glacial acetic acid and chloroform,
sparingly soluble in benzene, and insoluble in acetone and ether. They are
derivatives in which an aldehyde group is condensed with two hydroxyls of
glycerol to form an acetal (a gem-d\Q\htr). The third glycerol hydroxy 1 is
esterified with an aminoethanol-phosphate residue.

H

H— C— O H -X X

\|
C— R

H— C— O' O H H _.

HC— O— P — O— C— C— NHo

I OH I I ' --^.IL^-'

H H H

2. Phosphoinositides

These complex lipides containing inositol are numerous and little is
known about them.

3. Sphingolipides

In these complex lipides the alcohol is not glycerol but sphingosine,
a C18 aminoalcohol.

CH3— (CH,)io— CH=CH— CH(OH)— CH(NHo)— CH2OH

Sphingosine

{a) Sphingomyelins or phosphosphingosides — The pure crystalline sphin-
gomyelins are insoluble in ether and acetone, but soluble in benzene, hot
ethanol and hot ethyl acetate. They are emulsifiable with water.

76

UNITY AND DIVERSITY IN BIOCHEMISTRY

CH3

I

(CH,)i2

I
CH

II
CH

I
CHOH

H

HC N

^ Sphingosine

o

_ 11 T> } Fatty acid

HC

O

H

O

II

P O CH2-CH2 -N (CH3):

OH

OH

Phosphorylcholine
General formula of the sphingomyelins

(b) Cerebrosides — On hydrolysis these substances give sphingosine, fatty
acids and galactose. They are essential constituents of all cells (animal,
vegetable and fungi).

CH,

I

(CH2)i2

I

CH

II
CH

HOCH

HC-N— |-C— R
H

HC— O-
I
H

• Sphingosine

o

Fatty acid

O'

OH H H

I I I
C— C— C— C— C— CH,OH
I I I I I
H H OH OH H

Galactose
General formula of the cerebrosides

MODES OF LINKAGE BY COVALENT BONDS 77

III. PEPTIDE BOND

The peptide bond is an amide linkage resulting from the reaction of a
carboxyl group with an amino group, with the elimination of water.
Peptides are the result of joining two or more amino acids by the peptide
linkage. Example :

2 CH3— CH— COOH > CH3— CH— COOH + H2O

NH, NH— CO— CH— CH3

NH2

Alanylalanine
(a dipeptide)

A. Synthetic Peptides

The synthesis of peptides is of great interest, for, as we shall see, the
synthesis of an important natural polypeptide has confirmed the structure
assigned to it.

The most important synthetic method at the present time is that of
Bergmann and Zervas. It is based upon the fact that carbobenzoxy-
(CeHgCHaOCO-) derivatives of amino acids may be split by catalytic
hydrogenation.

Among other recent methods, we may quote the conversion of amino
acids into mixed anhydrides with carbonic acid; these latter compounds
react with an amino group to form a peptide bond. Similarly, carboben-
zoxy-amino acid anhydrides react readily with other amino acids.

B. Natural Peptides

(a) Glutathione, Anserine, Carnosine

Glutathione, a tripeptide found in animal and vegetable cells, has been
known for a long time.

REFERENCES

Celmer, W. D. & Carter, H. E. (1952). Chemistry of phosphatides and cere-

brosides. PhysioL Rev., 32, 167-196.
Kenner, G. W. (1951). The chemistry of nucleotides. Fortschr. Chem. org.

Naturstoffe, 8, 96-145.
Leloir, L. F. (1951). Sugar phosphates. Fortschr. Chem. org. Naturstoffe, 8, 47-95.
Singer, T. P. & Kearney, E. K. (1954). Chemistry, metaboUsm and scope of

action of the pyridine nucleotide coenzymes. Advances in Enzymology 15,

79-139.
ToDD, A. R. (1953). The nucleotides : Some recent chemical research and its

biological implications. Harvey Lectures, 47, 1-20.

78

UNITY AND DIVERSITY

IN BIOCHEMISTRY

HsC-

-SH

HC-

-CO-

-NH ^

O

. HN
CO

CH2 -
COOH

0

HgC

£

ca 'i
3

H^C

0

HC-

-NH2

. COOH

I

deduced

form ol

glutathione

Glutathione is a tripeptide made up of cysteine, glutamic acid and gly-
cine, it exists in two forms, reduced (or thiol form) and oxidized (disul-
phide form), or dehydrogenated glutathione.

HoC— S S CH2

NH— OC— CH

HC— CO— NH

CH2

I
COOH

HN
CO

H^C

!

i

H2C

NH

CO

I
CH2

CH2

HC— NH2 NH2— CH
COOH COOH

Oxidized form of glutathione

I
CH2

I

COOH

Carnosine or j6-alanyl-L-histidine is another natural peptide, present in
vertebrate muscle accompanied by its methyl derivative, anserine.

MODES OF LINKAGE BY COVALENT BONDS 79

COOH

NH,— CH,-CH,— CO— NH— CH-CHo— C=CH

HM Ts^

\//
CH

Carncsine

COOH
I
NH2-CH,-CH2-CO-NH-CH-CH.-C=CH

H3C— N N

\//
CH

Anserine

|8-alanine, which is present in carnosine and anserine, is not a consti-
tuent of proteins, but one finds it in other natural substances such as panto-
thenic acid, which is a dihydroxy-dimethyl-butyryl-/3-alanine.

CH3 OH

HO-CH2-C CH-CO-NH-CH2-CH2-COOH

1
CH3

Pantothenic acid

Interest in natural peptides has greatly increased during the last few
years since a great number of antibiotics have been found to be poly-
peptides.

(b) Antibiotics

Many antibiotics are peptides which are produced by microorganisms
and possess antibacterial properties.

The gramicidines produced by Bacillus brevis, for example, are cyclic
peptides having a molecular weight around 400, and containing chiefly
L-trytophane and D-leucine together with smaller amounts of D-valine,
L-valine, L-alanine, glycine and ethanolamine. One of the characteristics
of natural antibiotic peptides is that one finds in their structure amino acids
which are never present in proteins, or the D-stereoisomers of the natural
L-forms present in proteins.

80 UNITY AND DIVERSITY IN BIOCHEMISTRY

The tyrocidines are antibiotic peptides produced at the same time as the
gramicidines. Among their amino acids are L-ornithine and D-phenylalan-
ine. The penicilUns, produced by moulds of the genus Penicillium, are
derived from a dipeptide, a-formylglycyl-D-penicillamine.

CH3 CH3 CH3

\ / I

C— SH CHO H3C— C— SH

HoN— CH— COOH H2N— CH— CO NH— CH— COOH

Penicillamine a-formylglycyl-D-penicillamine

In penicillin G, for example, the penicillamine is cyclized into a thiazo-
lidine ring by reaction of the aldehyde group (formyl radical) with the
thiol and NH groups.

CHo -CO-NH CH CH C^

I I I

OC N CH

CHa

COOH

Penicillin G

In Other penicillins the benzene ring of penicillin G is replaced by other
groups.

(c) Phalloidin

The poison present in the fungus most commonly responsible for cases
of poisoning, Amanita phalloides, is a peptide known as phalloidin. On
hydrolysis, it gives cystine and alanine, but, in addition, allohydroxy-L-
proline, a diastereoisomer of the form of proline found in proteins.

{d) Peptide Hormones

The peptide type of structure is frequently employed for the chemical
transmission of messages by means of hormones. An example is provided
by the two hormones of the posterior hypophysis of vertebrates, oxytocin
and vasopressin. These two polypeptides have been extracted from the
gland itself, by rather a drastic treatment such that it still remains debatable

MODES OF LINKAGE BY COVALENT BONDS 81

OH

<
C

/ \
HC CH

II I

HC CH CH3

\ / I

C H2C CH3

1 \ /

CH2 CH

NH, O I O I

I II I II I

CHo-CH-C -NH-CH-C-NH-CH

I " Tyr lieu |

s c=o

I Cys I

S Asp (NH2) NH

I O O I

I II II I

CH2-CH— NH— C— CH— NH— C— CH— CH2— CH2— CONH2

I
C=0 CH2 Glu (NH2)

I I

I CONH2

I
H2C-N Pro O Leu O Gly (NH2)

\ II II

^ CH— C— NH— CH-C-NH-CHa-CONH2

/ I

H2C — C CH2

Hj I

CH

/ \

H3C CH3

Beef oxytocin

whether they circulate in the animal in the free or in the combined states.
Oxytocin and vasopressin are both octapeptides whose structures have been
confirmed by synthesis — no mean performance for a molecule of this
degree of complexity. This magnificent piece of work was carried out in
the laboratories of du Vigneaud in New York, Fromageot in Paris and
Tuppy in Vienna.

82

UNITY AND DIVERSITY IN BIOCHEMISTRY

OH
I H

c c

/\ // \

HC CH HC CH

II I I II

HC CH HC CH

\ // \ /

C C

I I

CH2 CHj

NH2 O O I

I II II I

CH2— CH-C-NH— CH-C— NH-CH

I Tyr Phe |

s c=o

I Cys I

S Asp (NH2) NH

I O O

I II- II

CH2-CH— NH-C-CH~NH-C-CH-CH2— CH2— CONH2

I

c=o

CH2

I

CONHa

Glu (NH2)

Gly (NH2)

HaC-N Pro O Arg O

\ II II

CH— C— NH— CH— C— NH--CH2— CONH,

/ I

H2C— C CH2

H2 I

CHa

I
CHa

I
NH

I
C

/ \
HN NHa

Beef vasopressin

REFERENCES

Bricas, E. and Fromageot, CI. (1953). Naturally occurring peptides, Advances in

Protein Chemistry, 8, 1-25.
Desnuelle, p. (1953). The general chemistry of amino acids and peptides, in

Neurath, H. & Bailey, K., The Proteins, Vol. I, part A, 87-180, Academic

Press, New York.
FRUTON,J.S.(1949).Thesynthesisofpeptides,^fift>a«cejmPro<«ViC/jewwfry, 5, 1-83.

CHAPTER IV

MACROMOLECULES

The knowledge of the various typical types of chemical structure which
have been identified in the biosphere, and of the principal linkages which
join them, still leaves us in a region where the essential identity of all
organisms may be'distinguished. There is no more difference between a
molecule of coenzyme A isolated from a bacterium and one prepared from
animal tissue than there is between two molecules of sodium chloride.
When covalency forces operate in a volume within the limits of a few cubic
angstroms to a few thousand cubic angstroms, we are still in the world of
simple molecules, or molecules joined together in the compounds described
in Chapter III : this is the region in which organisms are identical. This
truth has long intrigued biochemists, whose desire to understand the
chemistr}^ of life on a molecular scale has not prevented consideration of
the great diversity of living things. The advent of the chemistry of macro-
molecules introduced into biochemistry the idea of specificity, which up
till then was lacking.

Macromolecules are defined as chemical compounds whose molecular
weight is above 10,000 and in which covalent forces are effective in all the
available space. This more or less arbitrar}^ boundary corresponds approxi-
mately to molecular sizes above which the solution of these particles takes on
the so-called "colloidal" properties. But we are still dealing with chemical
molecules, even though these very large molecules cannot pass through
ordinary membranes. Their constituent atoms, like the compounds des-
cribed in Chapter III, are united mainly by covalencies.

As soon as one arrives in the world of macromolecular chemistry, one
must be careful to distinguish between the chemical molecular weight and
the physical molecular w^eight. The chemical molecular weight is the sum of
the weights of the atoms joined by covalencies, in the smallest particle of
that compound. The physical molecular weight is the weight of the particle
actually present in a gas or in a solution. An example, taken from Staudin-
ger, will illustrate this difference. The chemical molecular weight of
stearic acid CigHggOa is 284; the determination of the freezing point
depression in benzene reveals a physical molecular weight of 568. This
result is explained by the fact that the molecules of stearic acid, in which
the atoms are united by covalencies, are associated in pairs by the action of
residual valencies. The chemical molecular weight is certainly equal to

83

84 UNITY AND DIVERSITY IN BIOCHEMISTRY

284, for derivatives of stearic acid, such as the esters, contain the radical
CiaHggO-. (Radical = residue, group, grouping = aggregate of atoms,
which survive from one compound to another = residue of a molecule
when one or several atoms are removed. If an H atom is removed from
water H — O — H, the hydroxyl radical — OH remains. If an H atom is
removed from ammonia H — N — H, the amidogen radical — N — H
remains.) The idea of chemical molecular weight is derived from the idea
of a radical. When the chemical molecular weight is below 10,000 but is not
equal to the physical molecular weight, then one is dealing with molecules
associated in "micelles", as is the case with colloidal solutions of soaps.
When the chemical molecular weight, being above 10,000, is the same as
the physical molecular weight, then we are dealing with a solution of
macromolecules. When, however, the chemical molecular weight is above
10,000 but is less than the physical molecular weight then these molecules
are associated by residual valencies. In every case, the physical molecular
weight is either equal to, or greater than, the chemical molecular weight.

These polymers or macromolecules are made up of monomeric residues
by covalencies at two or more points. The natural macromolecules are
generally made up of long chains of such radicals joined by covalencies;
these chains may also be joined by a small number of side-chains, also
covalent in nature, such that the resulting structure takes the form of a
three-dimensional network.

The idea that proteins, cellulose, starch, etc., are polymers, that is that
they are made up of smaller units linked by covalencies, is not modern.
It dates at least from 1871, when the idea was clearly set out in a paper by
Hlaziwetz and Habermann. Unfortunately, these compounds were
classed by Graham among his "colloids", and there was for a long time
confusion between macromolecules and true colloids, in which the mole-
cules are linked by residual valencies. It was Staudinger who was respon-
sible for putting biochemists on to the right track once more when he
showed that the "colloidal" properties of solutions of macromolecules
persisted whatever the solvent, contrary to what is observed with micelles
resulting from the association of small molecules by secondary valencies.
Staudinger also demonstrated that the transformation of macromolecules
into their derivatives does not suppress their "colloidal" properties.

Among the macromolecules we find the same classes of organic com-
pounds as with simple molecules ; but, particularly important, the number
of isomers is very much greater with these larger molecules.

The chemical structures described in Chapters II and III have been
established by organic chemists, not only by means of analysis, but also
with the additional control furnished by synthesis. In the case of naturally
occurring macromolecules, synthesis is not yet possible for the chemist
(although Fraenkel-Conrat, after separating the nucleic acid from the

MACROMOLECULES 85

protein, has succeeded in recombining them to reform the macromolecule
of tobacco mosaic virus), so that it is not possible to say that any macro-
molecule in the biosphere is known in all its details. Nevertheless, the
study of synthetic polymers has greatly aided the understanding of natural
polymers.

I. POLYSACCHARIDES

Polysaccharides are very widely distributed in the biosphere, being
employed as a structural material (cellulose, xylan, chitin, etc.) and as a
form for storing the monomers (starch, glycogen, inulin, galactogen, etc.).

The polysaccharide molecule is formed by the association, by means of
oside bonds, of a large number («) of sugar molecules. The uronic acids
are associated into polyuronides in the same way as the sugars form
polysaccharides.

«(CeHi,Oe) - n H,o = (CeH,oO>

D-glucose cellulose

D-mannuronic acid
A. HOLOPOLYSACCHARIDES

[a) Polysugars or Polyoses
1. Hexosans

(a) Cellulose — The name "cellulose" is given to mixtures of homologous
polymers which give a quantitative yield of D-glucopyranose when hydro-
lysed in strong acid. Cellulose is the most abundant structural material in
plants. It is also found in many bacteria and even in certain groups of
animals, such as the Tunicates.

Cellulose is present in the pure state in the hairs of the cottonseed.
Complete acid hydrolysis of cellulose by strong, concentrated mineral acids
gives D-glucopyranose in quantitative amounts. Careful partial hydrolysis
in the presence of acetic anhydride and sulphuric acid (acetolysis) gives
molecules of cellobiose (4-D-glucopyranose-/3-D-glucopyranoside) and tri-
saccharides which can be hydrolysed by the enzyme emulsin. Hence, it

REFERENCES

Frey-Wyssling, a. 1957. Macromolecules in Cell Structure. Harvard University

Press, Cambridge, Mass., U.S.A.
Meyer, K. H. (1942). Natural and Synthetic High Polymers. Interscience, New York.
Staudinger, H. (1947). Makromolekulare Chemie und Biologie. Wepf, Bale.

86

UNITY AND DIVERSITY IN BIOCHEMISTRY

^ ,-^'8,35

Fig. 1 (Meyer and Mark) — The dimensions are shown in Angstrom units. The black
dots represent the oxygen atoms of the pyranose rings.

seems clear that cellulose contains only l:4-jS-linkages. Hydrolysis of
methylated cellulose yields 2:3:4:6-tetramethyl-glucopyranose and 2:3:6-
trimethylglucopyranose. Methylation followed by hydrolysis never gives
any dimethylglucose, so the chain must be a straight one. The chain
differs in length according to the source of the cellulose, and the values
obtained range between 1400 and 10,000 glucose units. The lay-out of
atoms is such that each cellobiose residue has the dimensions shown
in Fig. 5.

The cellobiose chains, arranged in the network illustrated by Fig. 1, are
grouped in bundles, the cellulose micelles, which are about 50A thick and
at least 500A long. The grouping of these micelles as they exist in structures
where the crystallites are parallel is shown in Fig. 2.

Fig. 2 (Seifriz) — Orientation of the micelles in a block of cellulose

MACRO MOLECULES

87

Sometimes, the micelles may be randomly oriented as, for example, in
cellophane (Fig. 3).

Fig. 3 (Mark) — Orientation of cellulose micelles in cellophane.

(b) Starch — The most abundant reserve of carbohydrate in plants and in
microorganisms is starch, which on hydrolysis is transformed quantita-
tively into D-glucose. In the starch molecule the glucose molecules are
associated by l:4-a-glucoside linkages. From most starches two constitu-
ents may be isolated :

(1) a straight-chain polysaccharide called amy lose which is coloured
blue by iodine. It consists of straight chains of variable length in which the
glucose units are linked by l:4-linkages and the number of units in each
chain varies from 100 to 2000.

Fig. 4 (Miller) — Different t>'pes of starch granule. A, from the haricot bean ; B, from maize ;
C and Ci, from potatoes (c, simple granule; Ci, composite granule); d and D^, rice grains
(d, whole of the composite grain; Dj, one of the constituents of the grain at a higher
magnification); E, wheat grain; F, composite grain from oats. Note that each elementary
granule (a, b, c, Ci, Dj, e) has a number of concentric striations around an initial point,

the hilum.

88

UNITY AND DIVERSITY IN BIOCHEMISTRY

(2) a highly branched polysaccharide, amylopectin, which is coloured
violet by iodine. The smaller side-chains are attached to the main branches
by l:6-a-linkages (isomaltose). The side-chains themselves, like the main
chain, are built up of l:4-a-glucoside linkages.

Certain starches contain only amylopectin. This is the case for the
starches from maize and rice.

When a starch is made up of amylopectin and amylose, the proportion
of the latter, like the length of its chains, varies according to the source.

CH.OH

CHtOH

CH.OH

CH.OH

Ot-

)H H OH

AMYLOSE

CH2OH

CHiOH

...O-

O H

O----

Branching by 1 : 6-a-linkages in amylopectin

(c) Glycogen — Glycogen, the major carbohydrate reserve in animals, is
a branched polysaccharide similar to amylopectin. It is made up of
D-glucose units. It is coloured brown by iodine and is water soluble
(15 to 20%). Glycogen is more highly branched than amylopectin and,
consequently, it contains a greater proportion of l:6-a-glucoside linkages.

MACROMOLECULES

89

Fig. 5 (Meyer and Bernfeld) — Structure of amylopectin.
o = a glucose residue. A = a reducing group. The dotted line shows the limit of
hydrolysis brought about by ^-amylase attacking the macromolecule at its surface.

(d) Other polyglucoses — One finds in the biosphere a great variety of
these ; a few are given below :

Lichenin from Hchens (straight chain; 30% of l:3-a; 70% of l:4-a)
Laminarin in the Laminaria (straight chain; l:3-j8; an average of 20
residues)

V o\y glwQosts oi Bet abaci erium vermiforme (l:6-a; 25 residues)

VoXyghicosts oi Phytomonas tumefaciens (1:2-/3; 22 residues)

Polyglucoses of the cellular skeleton of yeast (36 residues)

Polyglucoses of Leuconostoc mesenteroides (3-24 residues)

Polyglucoses of L6«co«05foc dextranicum (straight chain; l:6-a; 200-500
residues)

Fig. 6 (K. H. Meyer) — Structure of glycogen.

90

UNITY AND DIVERSITY IN BIOCHEMISTRY

(e) Galactans — They are frequently found in plants ; in the wood, in the
seeds and elsewhere. The galactan of lupin seeds is constructed on the
1:4-^ principle, and is made up of about 120 galactose units.

(/) Mannans — These are often present in wood, especially of coni-
fers, and they are difficult to separate out. The mannans of pine and
spruce are built of mannose molecules joined in straight chains of about
200 residues. The mannan of yeast, on the other hand, is highly branched
and is not homogeneous. The number of molecules of mannose in these
chains varies from 90 to 830.

(g) Fructosans (Levans) — The inulin found in the tubers of many of the
Compositae is a linear molecule containing about 30 D-fructose residues,
interspersed with about 6% of D-glucose units. Plants contain a multitude
of other fructosans (asparagosine, graminine, triticine, etc.) and so also do
bacteria. Unlike other fructosans, those in bacteria are highly branched
molecules.

2. Pentosans

(a) Xylans — Xylans are present in the lignified membranes of plants.
They are branched molecules made up of D-xylose units.

(6) Other pentosans — Plants contain many other types among which
araban may be mentioned.

{b) Polyuronides

The units here are uronic acids. The most numerous group is that of the
"pectic substances" or "pectin", in which the principal constituent is
pectic acid — a chain of D-galacturonic acid molecules united chiefly by
l:4-a-linkages. The pectins are not at all homogeneous and certain of their
constituents are still ill-defined.

Another polyuronide is alginic acid, present in marine algae and formed
of chains of about one hundred units of D-mannuronic acid, joined to-
gether by l:4-/8-glucoside linkages.

COOH H OH COOM ^ ^^

H OH COOH. H OH

COOH. H

Pectic acid (polygalacturonic acid)

MACROMOLECULES

91

(c) Polyglucosamines

Chitin, a constituent of fungi and of the exoskeleton of arthropods,
falls into this category.

Hydrolysis with boiling acids gives glucosamine and acetic acid in equiva-
lent amounts. A more careful hydrolysis with chitinase gives as the sole pro-
duct N-acetylglucosamine, that is, glucosamine acetylated at its amino group.

It has been possible to isolate from the products of a mild hydrolysis a
disaccharide, chitobiose, identical with cellobiose except that C-2 of each
glucose unit bears an amino group.

(»).

(b)

(a) Cellulose

CHjOH

(b) Chitin

R = CH3CO— (acetyl)

B. Heteropolysaccharides

{a) Gums and Mucilages

It is difficult to make any definite distinction between these two classes
of macromolecules. Gums, which exude from bark in the form of "gum
arable", are salts of heteropolyuronides. Mucilages, like that from linseed,
swell in water. In both cases we are dealing with highly complex branched
molecules containing several sugars. For example, in gum arable, arabinose,
galactose, rhamnose, glucuronic acid, etc., are all present, whilst in the
mucilage of the plaintain seed the following substances have been detected :
galacturonic acid, rhamnose, galactose, arabinose, xylose, etc.

(6) Mucopolysaccharides

These polysaccharides are invariably associated with amounts of protein
which, although they are small, are always present and are by no means
negligible.

1. Hyaluronic acid

This polysaccharide is very widely distributed, both in the free form and as
salt-like compounds with proteins. It is the inter-cellular cement in animals.

Hyaluronic acid is a complex polysaccharide containing equivalent
amounts of D-glucosamine, D-glucuronic acid and acetic acid combined
with glucosamine in the form of N-acetyl-D-glucosamine.

92 UNITY AND DIVERSITY IN BIOCHEMISTRY

2. Bacterial polysaccharides

(a) Pneumococcal — These polysaccharides control the immunological
type specificity of the pneumococci by their presence in the bacterial
capsule. Their constituents are D-glucose, D-glucuronic acid, aldobionic
acids and amino sugars. The proportions of each vary from one type to
another and certain constituents may be missing.

Examples of constituents which have been identified :
Type I. Galacturonic acid and an acetylhexosamine.
Type II. D-Glucose, D-glucuronic acid, L-rhamnose.
Type III. D-Glucose and D-glucuronic acid.
Type IV. N-Acetylhexosamine, D-glucose.

(b) Of other microorganisms — Mucopolysaccharides of numerous micro-
organisms have recently been studied. The luteose of Pencillium luteum,
for example, is a poly-D-glucose in which the glucoside bonds are of the
1:6-/3 type. The complex antigen has been isolated from the typhus
bacterium, Eberthella typhosa, it is a complex chain of sixty hexose units,
with 50% of D-glucose, 25% of D-mannose and 25% of D-galactose.
Tuberculin, the medium from the concentrated culture of Mycobacterium
tuberculosis, contains a polysaccharide in which there is D-arabinose, d-
mannose and D-galactose together with a little D-glucosamine. In the
mucopolysaccharide of Corytiebacterium diptheriae there are D-galactose,
amino sugars and pentoses.

Several mucopolysaccharides have been isolated from Penicillium
charlesii cultivated on D-glucose. Mannocarolose, one of these products, is
a mannan having a straight chain with 1:6 linkages, whilst galactocarolose,
from the same source, is a straight chain galactan containing 1:5 linkages.

3. Blood group polysaccharides

These are mostly polysaccharide in nature, but their molecules also
contain such substances as amino acids.

The substance of Group A, a branched molecule, contains L-fucose,
D-glucosamine, D-galactose and D-mannose. Very little is yet known about
the polysaccharides of the other blood groups.

C. Polysaccharide Sulphuric Esters, or Mucoitinsulphates

1. Heparin

Heparin is the sulphuric ester of a polysaccharide containing d-
glucuronic acid and D-glucosamine.

MACROMOLECULES 93

2. Chondroitin sulphate

This is the sulphuric ester of a polysaccharide whose main constituents
are D-glucuronic acid and N-acetylchondrosamine. The hyalin cartilage
of vertebrates is a compound of chondroitin sulphate and a protein, the
binding being between the -COOH and — SO3H groups of the chondroitin
sulphate and the -NHo groups of the protein.

3. Mucoitin sulphate

It is similar to the above compound except that chondrosamine is
replaced by D-glucosamine. It is present in many animal tissues.

II. PROTEINS

Proteins are macromolecules w^hich on hydrolysis yield a mkture of
amino acids. Whatever their origin, they are always made up of a selection
of the 20 amino acids described previously (p. 24). These acids are of the
L-configuration and joined together chiefly by peptide bonds. The various
properties of the proteins depend upon the number of amino acid residues
forming the peptide chain, on the nature of the amino acids, the order in
which they are assembled, the branching of their chains and on the con-
figuration of the folding which results from the free rotation of the parts of
the peptide chains about certain bonds.

A. Classification

Proteins are divided into two main types : fibrous proteins and soluble
or globular proteins. Most fibrous proteins are insoluble in aqueous sol-
vents. Although they do not crystallize, they contain crystalline regions.
They are formed from long molecules arranged more or less rectilinearly

REFERENCES

Evans, T. H. & Hibbert, H. (1946). Bacterial polysaccharides, Advanc. Carbohyd.

Chem., 2, 204-234.
Greenwood, C. T. (1952). The size and shape of some polysaccharide molecules,

Advanc. Carbohyd. Chem., 7, 290-332.
Kabal, E. a. (1956). Blood Group Substances. Academic Press, New York.
Manners, D. J. (1957). The molecular structxire of glycogens, Advanc. Carbohyd.

Chem., 12, 262-298.
McIlroy, R. J. (1948). The Chemistry of Polysaccharides, Arnold, London.
Mori, T. (1953). Seaweed polysaccharides, Advanc. Carbohyd. Chem., 8, 316-350.
Pigman, W. (1951). The Carbohydrates. Academic Press, New York.
Stagey, M. (1946). The chemistry of mucopolysaccharides and mucoproteins,

Advanc. Carbohyd. Chem., 2, 162-203.

94

UNITY AND DIVERSITY IN BIOCHEMISTRY

and more or less parallel to the axis of the fibre. The structural proteins fall
into this class. Their molecular weights are very high but hard to define
since they are generally insoluble.

By contrast, the molecular weights of the globular proteins are definite
(between 10,000 and several million) and their molecules are more or less
spherical. Most often they may be crystallized and they are soluble in
aqueous solvents (water or aqueous solutions of salts, acids, bases or
alcohols, depending on the particular protein). They may be denatured.
Active substances like enzymes, hormones, etc., belong to this class.

Fig. 7 (Springall) — Polypeptide ribbon in zig-zag form (^ form)

The fibrous proteins have many similarities to the synthetic polymers.
The latter are not very soluble and give infra-red absorption spectra and
X-ray diffraction spectra very similar to those obtained from the fibrous
proteins. The globular proteins do not resemble synthetic polypeptides.
However, complete denaturation transforms them into substances similar
to polypeptides.

(a) Fibrous Proteins

Organisms often employ fibrous proteins for supporting material. This
is most particularly the case in animals, for plants delegate the same
function preferentially to polysaccharides. Among the fibrous proteins are

MACROMOLECULES 95

the collagen of connective tissue, myosin of muscles, fibrin present in
blood clots, keratin and epidermin of vertebrate skin, the gorgonins of
coral, the conchiolines of mollusc shells, the sclerotins of the teguments of
arthropods, etc.

Apart from the determination of their amino acids, fibrous proteins have
chiefly been studied by X-ray diffraction methods. Astbury, applying the
results obtained by this method, classifies fibrous proteins into two groups :
the k-m-e-fgroup(keratin-myosin-epidermin-fibrin) and the collagen group.

X-ray diffraction spectra show that stretched fibres of keratin (/S-keratin)
are formed by the repetition of units 3-3 A in length, a figure very near the
calculated length (3-6 A) for the distance — NHCHCO — . The amino acid

R

Fig. 8 (Springall) — A sheet of polypeptide ribbons in zig-zag form (P form).

side-chains project on alternate sides of the main chain. The peptide
chains in j8-keratin are separated by a distance of 9.7 A. This distance is
near that calculated for the longest side chain of an amino acid such as
arginine (8-4 A).

Unstretched keratin (a-keratin) gives a different X-ray pattern. Fibrous
proteins of the k-m-e-f group give patterns similar to either that of a-
keratin or of /3-keratin, whilst the fibrous proteins of the collagen group
give patterns of another type.

The most generally (but not unanimously) accepted view is that the /3
form of the k-m-e-f group corresponds to a zig-zag structure (Figs. 7 and 8),
and the a form of the k-m-e-f group and the collagen group represents a
helical twisting, the axis of this twist corresponding to the fibre axis. The
most generally accepted helical structure is that in which there are 3-69
amino acid residues per turn (Fig. 9). In certain fibrous proteins the

96

UNITY AND DIVERSITY IN BIOCHEMISTRY

polypeptide chains with the a-helical structure are arranged in parallel with
each other. In other proteins they are themselves associated in threads in
which the polypeptide chains are twisted together (Fig. 10).

(b) Globular Proteins

A profound study of the many globular proteins has been carried out
over the last few years, information being obtained from the study of osmo-
tic pressure, diffusion, viscosity, sedimentation, electrophoresis, light

Fig. 9 (Pauling, Corey and Branson) — Portion

of a-helix.

N = nitrogen atom; R = side chain; black dots

= hydrogen atoms ; blank circles = carbon atoms ;

dotted circles = oxygen atoms; dotted lines =

hydrogen bonds.

diffraction, birefringence of flow, dielectric properties, infra-red absorption,
X-ray diffraction and by the use of the electron microscope. This imposing
array of techniques has provided an immense amount of data the systemiza-
tion of which remains almost impossible. Certain general conclusions,
however, may be drawn from these results.

MACRO MOLECULES

97

(a) Molecular weights

The molecular weights of globular proteins lie between 10,000 and
several million. Several attempts to arrange these molecular weights into
groups (Svedberg; Bergmann and Neumann) have not stood the test of
time, and one is forced to admit that the laws governing the molecular
weights of proteins are still unknown to us.

Fig. 10 (Pauling and Corey) — Forms in which
a-helical polypeptide chains may be twisted to-
gether. An ABg bundle (six chains rolled around
a seventh : this is the structure of the keratin
found in hair and nails) or a D3 cord (three
rolled chains).

50A

(a)

(b)

(b) Shape

This is sometimes visible in the electron microscope, as is the case for
haemocyanin (Fig. 11). In other cases, measurement of physical constants
permits the dimensions of the protein molecule to be calculated approxi-
mately. Thus, beef insulin (M.W. = 12 X 10^) is a right prism 44 A long,
26 A wide and 20 A thick, whilst tobacco mosaic virus (M.W. = 4 X 10')
is a rod 2980 A long by 150 A in diameter.

(c) Structure

From the sum total of the evidence it appears that the globular proteins
have a structure similar to the a form of the k-m-e-f group of the fibrous
proteins, that is, a helical twist with 3-6 residues per turn or 18 residues per
5 turns. However, this does not explain the compactness of the globular
proteins. It appears that the polypeptide ribbons are bunched into compact
globules and held by lateral linkages between the chains.

H

98 UNITY AND DIVERSITY IN BIOCHEMISTRY

The flexibility required for this bunched state, as Neurath has suggested,
may be obtained by the presence of an amino acid such as glycine, which is
without side chains to prevent free rotation. Or, in Pauling's opinion,
proline may be a point of flexibility since, when part of the peptide structure,
it does not possess an NH group, does not form hydrogen bonds
N-H . . . O, and retains full liberty of bending.

H HoC CHo

---N I I

\ CH CH2

CO N

H I

N CO

/\/
- - - CO CH

I
R

Diagram showing the possibility of a change in direction of the
peptide chain at a proline residue.

id) Denaturation

The globular proteins can undergo denaturation, a process which is
often irreversible, but is not well defined ; it may result from the action of
many and diverse agents (urea, alcohol, detergents, ultrasonic vibrations,
etc.) and is revealed by a decrease in solubility, an increase in viscosity, the
appearance of free — SH groups, etc. Denaturation is characterized by a
very high temperature coefficient, which implies a high degree of order in
the molecule of native protein.

On the other hand, the enthalpy ( — zJH, heat liberated at constant
pressure) is low, indicating little change at the level of covalent bonds.
Denaturation appears to be a sort of collapse of a highly ordered and specific
arrangement. The result is a poorly ordered mixture of polypeptides.

All these observations lead us to consider a globular protein as a highly
ordered three-dimensional structure of polypeptide layers of the a-k-m-e-f
configuration (probably containing 3-6 residues per turn) bunched into a
globular mass and maintained thus by linkages between side-chains and
by relatively weak hydrogen bonds. In the region of a polar group having
a spare hydrogen atom, there is an attraction for the negative charges of
neighbouring molecules. In two neighbouring peptide chains, a peptide
hydrogen may form a bridge with a pair of electrons on an oxygen atom of
the other chain.

ri

J"

1,.*-^ ■

^ii'-^

.<*..

^'■r

I-

r-;

*•

• j. <- .4«*. ■ aS

• 4 '

.■/.> ,

l7^

*-•'■'- .^ '

"'

■' ' :^.

/.'j!

1 *

^; ■

v*i

"^r J-

%

'^ M .

■■^%

t ^ ll— 1

Fig. 11 (Keith R. Porter) — Haemocyanin from Liinulus. The spherical
particles are the haemocyanin molecules slightly Hattened by drying.

MACROMOLECULES 99

R O H R O H R

: i: H I I II H I I

C C I N C C I N C

\/;\/\l/\/l\/\l/\/\/

CjN C C|N C C N

.i H I I II H I 1 II I

O H R O H R OH_H

HHO R H O R H O

I II I H I H II I I H II
N C C|N|C C N|C

/ \l/ \ / \l/ \ / \ /1\ / \|/ \

C N C C N I C C

i I II I I H 11 I

R H O R H O R

• • • •

• • • •

The formation of hydrogen bridges is an expression of the tendency
possessed by hydrogen atoms to share the electrons of an oxygen atom.

V • • • • \. • • • •

c::o: h:o— -* c:;o:h:o —
/ ,. .. / •• ••

Hydrogen b onds may also be formed between hydrogen and nitrogen atoms
between -OH of tyrosine and free -COOH groups, and also between amides

\ /

N-H H 0=C

0=C 0---H— N N-H

\ '^ \ /^

H-C-CH0-CH2-C C-CH2-C— H

/ ■ \ / \

H— N N— H---0 C=0

\ I /

n H-N

\

Glutamine Asparagine

Other bridges between polypeptides are of the disulphide type (covalent),
or of a salt-like nature (non-covalent, electro-valent).

We have said that the shape of the globular proteins depends upon
relatively weak secondary bonds. On raising the temperature, thermal
agitation of the molecules may be sufficient to break these secondary bonds.
Mineral salts and urea, by polarizing the water molecules around their
molecules or their ions, cause dehydration of the globular macromolecule
thus modifying the electrical field of force around it and changes in shape
result. Acids and bases, by modifying the ionization of basic and acidic

100 UNITY AND DIVERSITY IN BIOCHEMISTRY

groups, also modify the electric field and the shape of the macromolecule.
Heavy metals, which form coordination complexes with certain groups,
act in the same way. In many cases this denaturation and change in shape
is accompanied by a tendency to pass into a fibrous state and at the same
time certain functional groups which were hidden in the interior of the
molecule are revealed. The uncovering of these groups certainly plays a
part in the tendency displayed by denatured proteins to form aggregates.
The formation of these aggregates and the accompanying decrease in
solubility are facilitated at the isoelectric point since then nothing prevents
the molecules from coming together.

Denaturation may be irreversible. This is the case when important
changes in structure have taken place altering the geometry of the electric
field of the globular molecule.

/

\

o=c

C=0

\

/

C-CHs-

-s

-S—

CHs-C

/

\

H-N

N-H

\

/

C=0

0 = 0.

/

\

R-C~H

H-C-R

\

/

N-H

H-N

/

\

0=0

C=0

\

/

H-C-CHa-

-CHs-

-coo-

.-•NH,-

-C-

NH-

-CH=-

-CH2-

-CHi-C-H

,,^/ Glutamic acid

II

Ai'gininc \

HN

NH

c=o

\

/

C=0

/

Irreversible denaturation may be accompanied by polymerization and a
fall in solubility. The appearance of a precipitate is called flocculation; if
the precipitate is practically insoluble it is called a coagulum.

Denaturation may be reversible, if the changes in structure are slight.
When the initial conditions are restored, the electrostatic field reestablishes
the original shape of the globular molecule.

B. The Nature and Positions of the Constituent Amino Acids in

THE Protein

{a) Nitrogen and Sulphur Content

One of the present tasks of biochemistry is the determination of the
order in which the amino acids are assembled in the protein polypeptide
chains and the description of the structure of that protein. It is first of all
necessary to know the total protein nitrogen (around 16%) and the total

MACROMOLECULES 101

sulphur (around 2%); when the different amino acids have been deter-
mined these figures allow one to check that the sum of these amino acids
accounts completely for the composition of the protein.

(b) Titration

The reactive groups of the protein may be determined by titration, with
acids and dilute alkalies when dealing with acid or basic groups, and by
means of silver nitrate, iodine, etc., when dealing with thiol groups.

The potentiometric titration of a protein in aqueous solution with an acid
or dilute alkali is carried out by measuring the change in pH which results
from the addition of a known amount of the acid or base to the isoionic
protein. The isoionic state exists when the number of protons attached to
the basic groups (e.g. -NHg + H+ -> -NH+g) is equal to the number of
protons removed from the acidic groups (e.g. -COOH -^ -COO" + H+).
If there are no other ions apart from protons fixed on to the protein,
then the isoionic state coincides with the isoelectric state, defined by charge
O. The results of the titration are expressed in terms of change in pH/unit
of acid added and change in pH/unit of base added. Figure 12 shows the
dissociation curve of the protein.

There are quite a number of ionizing groups present in proteins :
a-COOH the terminal group of polypeptide chains;
^-COOH or y-COOH of aspartic or glutamic acid;

n NH of histidine;

N

H

a-NH+3 the terminal group of polypeptide chains;
€-NH+3 of lysine;

-<^ ^-OH of tyrosine;

SH of cysteine ;

---NH

\ + . • •
C=NHo of argmme.

/

H2N

When the pH is very acid, around 1-0, for example, all these groups are in
the undissociated state. At pH 14-0, they are all completely dissociated.
As one passes from pH 1-0 to pH 14-0 each ionizing group will dissociate

102

UNITY AND DIVERSITY IN BIOCHEMISTRY

over a particular pH range corresponding to its pK value. Figure 13
illustrates this. In Fig. 12, three regions, labelled a, b and c, may be dis-
tinguished, in which a slight change in pH has had a marked effect on the
number of protons combined with the protein. Referring to Fig. 13, we can
see that region a corresponds to the dissociation of terminal free-carboxyl
groups, and region h to that of the dissociable groups of histidine and
terminal a-amino groups. Region c corresponds to the dissociation of the
groups of lysine, tyrosine, cysteine and arginine. To investigate the ques-
tion further and resolve the complexities of regions b and c it is necessary

pH-*

Fig. 12 (Springall) — Titration curve of a protein. Abscissae : pH values. Ordinates :
the number h of protons added or subtracted, starting from the isoionic state, to give these
pH values in aqueous solution. These values of h are obtained from the number of equiva-
lents necessary to bring the solvent to the same pH.

to mask one or other of the groups. Thus, free amino groups may be re-
acted with formol or removed by enzymatic deamination, etc. The com-
plete and often arduous analysis of the titration curve of a protein gives us
information about the amount of arginine, histidine and lysine in that
protein, and also the total number of free carboxyl and a-amino groups.
Knowing the number of primary amide groups (from the amount of
ammonia in the hydrolysed protein — see later) and the amount of aspartic
and glutamic acids, it is possible to calculate the number of carboxyl
groups not involved in peptide bonds. If the number of free carboxyls and
the number of free amino groups are known some idea may be obtained of
the number of polypeptide chains present in the protein molecule.

{c) Composition of the Hydrolysate

When the titration of the native protein has been carried out the amino
acid composition is determined. Hydrolysis gives a mixture of amino

MACRO MOLECULES

103

acids and ammonia. The latter derives from the amide groups of glutamine
and asparagine, but it may also be an artefact arising from the breakdown
of certain amino acids during the hydrolysis. Since the average molecular
weight of the amino acids is 110, then complete hydrolysis of a protein of
molecular weight 36,000 will produce around 300 molecules of amino
acids of 20 different kinds. It is evident that the analysis of this hydrolysate
is a formidable problem whose solution was a notable achievement. The
methods used are numerous : specific precipitation of some groups of
amino acids (for example by phosphotungstic acid in the case of cystine,
arginine, histidine and lysine); precipitation of the amino acid directly

pH-*
2 3 4 5 6 7 8 9 10 U !2 13

[---R-HF*
-COsH(a,p,Y)

NH

J

NH
-'^fH3(a)

-*NHa (8)
-^>0H

-SH

NH.

^C=*NH,
-NH

^^

^^^^^

b

h

^

»

^

^

[-..R]ln->l*+H*

-cor

N

J

NH
NH.(a)

-NH-le)

-s-

NHj

\irNH

-NH

Fig. 13 (Springall) — Dissociation constants of the different groups — R-H. The dashed
areas indicate the pH range in which dissociation takes place when the pH increases.

(proline by ammonium rhodanilate) ; colorimetric methods; isotope dilu-
tion methods; enzymatic methods (for example, measurement of COg
liberated by a specific decarboxylase); microbiological methods — depend-
ing on the fact that for a certain strain a certain amino acid is indispensable
for growth, the latter may be measured (for example, in the lactic acid
bacteria by the production of acid) and is proportional to the concentration
of the amino acid; chromatographic methods, etc.

Table IX shows the amino acid composition of a collection of proteins
whose analysis is complete or almost complete. As can be seen, such a table
does not permit any useful conclusion to be drawn from it. Thus attention is
focussed on the sequence of the amino acids in the protein polypeptide chains.

104

UNITY AND DIVERSITY IN BIOCHEMISTRY

(d) Determination of the Amino Acid Sequence

A number of methods allow the removal of an amino acid residue from
the N-terminal end of a polypeptide terminal residue :

+ I
NH,-CH-CO--

R

Such a method is that of Edman (1950) in which phenylisothiocyanate
in pyridine at pH 9-0 is used. The principle is as follows :

NCS NH,CHCO— pept.
A R

V

+

Pyridine

NHCSNHCHCONH— pept.
/\ R

V

Phenylisothiocyanate

Phenylthiocarbamylpeptide (PTC-pept.)

Anhydrous HCl

V

N-CS

CO NH

\ /

CH

I

R

+NH2 — pept.

R

Alkali

- NH,-CH-COOH

N-terminal amino acid

The phenylthiocarbamyl derivative of the peptide when treated with
anhydrous HCl gives the phenylthiohydantoin of the N-terminal amino
acid which, when treated with Ba(0H)2 in alkaline solution, liberates this
terminal amino acid.

This method is applied to the polypeptides obtained from proteins by
various means. It is possible to remove the N-terminal amino acids one
after the other and identify them while still keeping the rest of the chain
intact for a further shortening and further identification of the amino acids
obtained. There are also methods which allow amino acid residues to be
chopped off from the c-terminal end of the chain.

To obtain these polypeptides from the protein, it may be hydrolysed
carefully with a 10 n mixture of hydrochloric and acetic acids for several
days at 37°, or hydrolysed with alkali or by the action of enzymes.

A witness to the success of these methods is the determination, by
Sanger, of the amino acid sequence of a part (fraction B, one of the two

MACRO MOLECULES

105

S

«o

St

Sf

•Ki

's*

o

>«i

;k

O

•c^

in

^

o^

^

R

«

o

O

" \

T— 1

cs

t^

a

"tl

ifc

yi

a

S

o

U4

c;

ft:

• f*A

X

c

S

►— 1

'ci

M

■^

J

^

.S

uout x/Cqmofi

oo

o
m

o

00

'I-

6

<N

1

1 1

1

NO

tn

tN

00

6

tN

4

in

in

tn

6

in

tN

00

i^

o

m

ON

m

00

00

in

■+

•+

NO

r^

'I-

tN

uisoXuiodojjL

1 00

1 Ov

o>

1

^~'

tN

NO

1 2

O

in

o

00

nO

(N
tN

5^

tN

t^

ts

00

t^ in

•<*■

<N

r^

in

ON

o

m ■^

o

00

^

o

in

t^

m

r^

m

uaSouuqij

?:5

in

in

NO

ON

tN

ON

n r^

vO

5

tn

NO

00

ON

00

ON

nO
NO

in

in

NO
'I-

00

O fN

O

o

t^

rj

00

nD

«— 1

o-

o

in

o

On

m

00

(N

t^

auiXzosAq

o in

♦t
■*

in

On

(N

00

1-H

NO
NO

1 2:

in

nO

ON

NO

ON

tN

NO

NO

00

m

00

o 00

■*

(N

t^

ON

NO

-o

^

OO

r^

r^

(Zr.

o

NO

t-4

On

NO

asEiXJoqdsoiidaQ

in in

(N

so

O
00

On

o

(N-)

tn

1 2

On

NO

-o

tN

ON

tN

ON

NO

tn

NO

tn

tn

rNl

00

3SBua3ojpXqap

ajEqdsoqdasouj^

— Th
-^ in
00 r-«

o

ON

m

On

NO

o

tn

ON

1 t

O
6

6

tN

NO
NO

ON

tn
rt

r~

s

in

On
00

o to

fS

t^

f)

t^

in

tn

On

<ri

■*

rNl

■+

o

t>

m

ON

tn

9SBIOP1V

CO

VO

00

o

NO

ON

00

ON

1 ^

-

nO
tn

tN

m

NO

On

NO

tN

NO

On
tN

O
tN

On

m

vO

m

en

■*

t^

nO

-. ^

NO

t^

00

tN

<N

ON

„

NO

On

uisdaj

00 1

o

ON

<N

00

00

to

r^

-i- --

*"*

in

m

NO

O

CNl

On
00

nO

o

00

NO

On
00

o

<N

m

in

n

00

in

t^ tN

tn

ts

On

I^

00

CnI

in

t^

r^

uaSouisdXJjotuXq^

•=> 1

vO
00

ON

r-l

in

IN

tN

O X

tN

NO

t^

1^

in

00

NO

00

o

in

On

NO

00

ro

ro

nO

f-)

00

(N

r^

NO

tN

^

t

r^

rNl

nO

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UNITY AND DIVERSITY IN BIOCHEMISTRY

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MACROMOLECULES 107

polypeptide chains joined by disulphide bridges) of the insuhn molecule.
Table X shows the amino acid sequence, as determined by Sanger, in a
series of polypeptides separated from fraction B of insulin. Sanger has
demonstrated that the only possible sequence corresponding to these many
peptides is that shown at the bottom of the table.

III. NUCLEOPROTEINS

The nucleoproteins are macromolecules formed by the union of proteins
and nucleic acids. The nucleic acids themselves are polymers of nucleo-
tides. The complete hydrolysis of the nucleic acids separated from nucleo-
proteins gives phosphoric acid, purines and pyrimidines (six members of
these two groups have been identified up to the present time), and two
furanose sugars. These latter are, either D-ribose (ribonucleic acids) or
2-desoxy-D-ribose (desoxyribonucleic acids). They have either two or
three -OH groups available for esterification. The number of possible
nucleotides entering into the composition of nucleic acids is, therefore, very
great. The purines, adenine and guanine, enter into the composition of all
nucleic acids. Among the pyrimidines, cytosine is present in all the nucleic
acids. Besides these purines and the pyrimidine which are always present,
one finds uracil in ribonucleic acids, and thymine and 5-methylcytosine
in desoxyribonucleic acids. Analysis of the nucleic acids reveals that they
are polymers of nucleotides. However, we are still far from knowing the
nature of the complex mixture of polynucleotides which make up each
nucleic acid. The little that is known at the present time is due to a com-
bination of the results of chemical hydrolysis and step-wise degradation by
means of enzymes.

A. Properties of the Two Types of NucLEit Acid

(a) Ribonucleic Acids (RNA)

These complex mixtures contain only four bases : adenine, guanine,

cytosine and uracil. The relative proportion of each of these bases does not

differ appreciably from unity. Considering that this is not the case for the

REFERENCES

Edsall, J. T. (1958). Aspects actuels de la biochimie des acides amins et desprotemes.
Masson, Paris.

Fox, S. & Foster, J. F. (1957). Protein Chemistry. Wiley, New York.

Neuberger, a. (1958). Syjnposiiim on Protein Structure. Methuen, London.

Neurath, H. & Bailey, K. (1953-1955) The Proteins. Chemistry, Biological
Activity, and Methods. (A collaborative work by many specialists in this field,
it is both the most comprehensive and the most modem text book available.)
4 vols. Academic Press, New York.

Springall, H. D. (1954). The Structural Chemistry of Proteins. (More concise
than the above, it gives a clear and constructive account of the methods and
results of the chemical study of protein structure.) Butterworths, London.

108 UNITY AND DIVERSITY IN BIOCHEMISTRY

polynucleotide chains isolated from ribonucleic acids, this is an indication
of the complexity of what is customarily called a "ribonucleic" acid. The
nucleosides entering into the composition of the ribonucleic acid from
yeast are /3-D-ribofuranosides in which adenine and guanine are linked at
N-9 and cytosine and uracil at N-3. It appears that ribose is always the
sugar present in what were formerly known as pentose-nucleic acids and
they can, therefore, be named ribonucleic acids.

The internucleotide bond which is predominant in the ribonucleic acids
is the phosphoric ester bridge between C-3' and C-5' of two adjacent
nucleotides.

Cs— OH Ca—OH C=— OH C3-OH

I I i I

Cs.-O OH Cs'-O OH Co— O OH C.—O OH

\ /
P

\ / I \ / I \ /

P P P

// \ // \ ! // \ I // \

_C,, O O C.. O O C.. O O C5' o o

In such a scheme, — C-2' — C-3' — C-5' — represents a nucleotide residue.
The presence of an -OH on C-2' explains how, by analogy with the results
obtained with mononucleotide esters, the alkaline degradation proceeds
through the intermediate formation of a cyclic structure in which nucleo-
side-2', -3' cyclic phosphates are formed by cleavage of the C-5' — O — P
bond, as shown above.

c,' — o •'" o Ca — o ..-■ o

Cv

— 0 ,0 c,-

— 00

^^£

\ -^ ^

^r.'^

1

p /

P

1

/ X '

y \

Ca-

— 0 / 0 c.

0 0

1

\;

\

Cv

Q.

The isolation of ribonucleic acids is difficult which makes the determi-
nation of their structure and composition difficult also. However it can
be shown that the ribonucleic acid of animals differs from that of yeast, and
that the ribonucleic acids from different organs of the same species are less
alike than are those from a given organ obtained from several species.
Unlike the desoxyribonucleic acids, the ribonucleic acids differ not only
from species to species but also from tissue to tissue in the same species.
Moreover, the ribonucleic acids of the nucleus differ from that of the
cytoplasm, and external conditions also cause variations.

(b) Desoxyribonucleic Acids (DNA)

The desoxyribonucleic acids which do not have an -OH group on
C-2', differ from the ribonucleic acids in possessing a more stable inter-
nucleotide bond. Although the linkage is between C-3' and C-5' of adjacent
sugar molecules, the cyclization described above for the ribonucleic acids

MACRO MOLECULES

109

is impossible, which gives the desoxyribonucleic acids a very great stability.
Their general structure is the following :

Base

i

\

\

Base

\

P

\ \

Base

'%'

Base

■■z'

\ 1 \
p i p

\l \

'5'

C,/

Q'

The concept of the helical structure of the desoxyribonucleic acids was
first suggested by Pauling and Corey (1953), following research by the
X-ray diffraction method. A helical structure which accounts more fully
for the experimental facts has been proposed by Watson and Crick (1953)
(Fig. 14). It consists of two helical chains rolled around the same axis.
The two chains are twisted in parallel but the order of the atoms is inverse.
The purines and pyrimidines are directed to the interior and the phos-
phate to the outside. Along the helix a nucleotide occupies a distance of

34 A

i

3.4 A

Fig. 14 (Watson and Crick) — Helical structure of desoxyribonucleic acid.

no

UNITY AND DIVERSITY IN BIOCHEMISTRY

3*4 A, and the repeating unit which is made up of ten nucleotides occupies
34 A. The angle between two adjacent nucleotides in the same chain is
36°. The purine and pyrimidine bases, being directed to the interior, are
perpendicular to the axis of the helix. They are associated in pairs, one
from each chain by means of hydrogen bonds.

As there is' not enough room to allow two purines end to end, and since
two pyrimidines would form too short a bridge, the only possible linkages

Adenine

Thymin*

Guanine

Crlo«<nt

Fig. 15 (Watson and Crick) — Hydrogen bonds compatible with the formula of Watson

and Crick.

are adenine-thymine and guanine-cytosine linkages. The shape of these
links is represented in Fig. 15.

The desoxyribonucleic are more stable than the ribonucleic acids and,
consequently, a greater number of results have been obtained in deter-
minations of their molecular weight. They are very elongated, threadlike
molecules, whose molecular weight is in the region of six million.

The composition of the desoxyribonucleic acids varies from one species
to another, but their composition appears to be the same in different organs
and does not appear to be influenced by the environment.

MACROMOLECULES 111

B. The Nucleoproteins

The nucleoproteins of the nuclei of trout, salmon and herring sperm, are
made up almost entirely of protamine and desoxyribonucleic acid. The
nucleoproteins of thymus are made up of desoxyribonucleic acid (40%),
ribonucleic acid (1-2%), histones and non-basic proteins. The cellular
nuclei contain chiefly desoxyribonucleic acid but, in addition, there is a
little ribonucleic acid (in the nucleolus and chromosomes). The cytoplas-
mic nucleoproteins in general contain only ribonucleic acid associated
with proteins which do not have the basic properties of those joined to
desoxyribonucleic acid. But the reproductive cell of animals (oocytes)
contain desoxyribonucleic acid in the cytoplasm. The desoxyribonucleic
acid of the chromosomes is combined with histones, protamines, and a
protein of the usual type, which is referred to as residual protein.

Protamines not possessing primary amino groups are associated with
DNA by a salt linkage, and this is also the case for the histones. Separa-
tion takes place when an extraction is made with solutions of high ionic
strength. The protamines and histones are very heterogeneous. The
residual protein, unlike the histones, contains tryptophan.

IV. METALLOPROTEINS

The metalloprotein structure is one which is widely and diversely
employed in the chemistry of living cells.

A. METALLOPROTEINS IN WHICH THE MeTAL IS BoUND TO THE PrOTEIN

Through the Intermediary of Another Structure

(a) Haemoproteins

Iron possesses 26 electrons distributed among 4 shells (K, L, M, N).
The K and L shells are saturated and contain 2 and 8 electrons respectively,
all of which are paired ; the N shell contains 2 paired electrons ; the M shell
contains 14 electrons. The M shell has 1 5 orbital, 3 p orbitals and 5 d
orbitals; the energy level of the 5 orbital is below that of the ^ orbitals which
is less than that of the d orbitals. Each orbital can contain 0, 1 or 2 elec-
trons; in the last case the two electrons have opposite spins and their
magnetic effects cancel out (paired electrons). First of all, the electrons

REFERENCES

Bracket, J. (1952). Le role des acides nucleiques dans la vie de la cellule et de I'embryon,
Masson, Paris and Liege, Desoer, (Actualites biochimiques, No 16).

Chargaff, E. & DAvmsoN, J. N. (1955). The Nucleic Acids. Chemistry and Biology.
2 vols. Academic Press, New York. (The collective effort of leading workers
in this field. This work contains full information and a constructive discussion
of the subject.)

112 UNITY AND DIVERSITY IN BIOCHEMISTRY

fill the orbitals which have the lowest energy level, so that in iron, the s and
p orbitals of the M shell are saturated, making 8 electrons in all (4 pairs).
The 6 remaining electrons are in d orbitals and they tend to occupy as
many of these as possible : one orbital contains two paired electrons and
the four others each contain one unpaired electron. So iron possesses 4
unpaired electrons.

Each unpaired electron, on account of its spin, behaves as a small
magnet : it becomes oriented in a magnetic field so as to oppose the
field. Measurements of magnetic susceptibility allow the number of un-
paired electrons to be determined. Substances which, like iron, contain
unpaired electrons are said to be paramagnetic and those which contain
only paired electrons are called diamagnetic.

The valency electrons are usually those in the outer shell. However, in
the case of iron, the energy level of the d orbitals of the M shell is almost the
same as the energy level of the s orbitals of the N shell, so that the d
orbitals of the M shell take part in the formation of iron complexes.

The iron atom loses two electrons and becomes a ferrous ion; the two
electrons which are lost are two paired electrons of the 5 orbital of the N
shell; the ferrous ion then, like iron itself, contains 4 unpaired electrons.

The loss of an additional electron to give the ferric ion is at the expense
of the six electrons in the d orbital of shell M ; the 5 remaining electrons
redistribute themselves among the 5 d orbitals and ferric iron contains
5 unpaired electrons.

In ferrous or ferric coordination complexes containing covalent links,
the complexing groups bring two electrons per bond. Four electrons can
be introduced into the d orbitals of the M shell (saturating them in the case
of ferrous complexes and leaving one unpaired electron in the case of ferric
complexes) and 8 other electrons will be required to saturate the 5 and p
orbitals of the N shell, conferring upon this shell the very stable octet
structure. It can be seen that the coordination number of the ferrous ion, as
well as that of the ferric ion, is 6 : two bonds established through the M shell
{d orbitals) and 4 bonds through the N shell {s and p orbitals) of the ion.
These hybrid bonds (hybrid, because two electronic shells are involved) are
denoted by the symbols d^sp^. The complex has the form of an octahedron
with the centre occupied by the ion ; the coordination linkages which are
covalent (2 shared electrons per bond) are directed towards the 6 corners.

1. Haems

The porphyrin molecule can combine with heavy-metal ions such as
ferrous, ferric, cupric ions, etc.

Haem refers to the ferro-porphyrin which results from the combination
of a porphyrin with a ferrous ion :

porphyrin + Fe++ 5± ferro-porphyrin + 2H+

MACRO MOLECULES

113

It is obvious that the reaction is pH-sensitive, in acid solution the
compound is completely dissociated. The loss of the two protons from the
porphyrin molecule leaves behind two negative charges which, by means of
the system of conjugated double bonds, are distributed by resonance over
the four nitrogen atoms.

The ferrous ion is paramagnetic ; it has 4 unpaired electrons. Likewise,
the haem molecule is also paramagnetic and measurements of magnetic
susceptibility have shown that, like the ferrous ion, it has 4 unpaired
electrons; this shows that the nitrogen atoms of the porphyrin bound to
the ferrous ion do not share any electrons with it; the bond is ionic. In the

CH

CH,

COOH GOGH

Protohaem

ferro-porphyrin complex, the ferrous ion has a coordination number of 6 :
in the complex, it retains two water molecules by means of ion-dipole bonds.
The 6 bonds of the complex are, therefore, electrostatic in nature.

Ferro-porphyrin can be represented :

N-

N-

Fe++

N-

or

N --

N

I
I

I
I

I /
— Fe -

H2O

N

H,0

I

N

2. Haemochromogens

The water molecules in the ferro-porphyrins are readily replaced by
nitrogen bases; the name "haemochromogens" is given to the new complexes
so formed.

114

UNITY AND DIVERSITY IN BIOCHEMISTRY

This substitution reaction is accompanied by a complete rearrangement
of the structure of the complex. Measurements of susceptibiHty now indi-
cate that the haemochromogen no longer possesses any unpaired electrons :
it is diamagnetic. The two molecules of nitrogen base and the four por-
phyrin nitrogen atoms have shared their electrons (12 in all) with the ferrous
ion; there are no remaining unpaired electrons and the 6 coordination
bonds of the complex are covalent (d^sp^). The haemochromogen has the
shape of an octahedron with the ferrous ion at the centre and the nitrogen
atoms at the 6 corners. The 4 nitrogen atoms of the porphyrin molecule are
in the same plane. The structure of the haemochromogens is exactly the
same as that of the ferrocyanide ion, Fe (CN)6, which is also diamagnetic.

A haemochromogen can be written thus :

N

NR

N

/

h-

Fe

^'

•- I

/

,--''

1-

S

— N

N

3. Haematins and parahaematins

Oxidation of the ferrous ion of haem gives rise to a ferri-porphyrin which
is given the name "haematin". An oxygen molecule can bring about this
oxidation.

The ferric ion has 5 unpaired electrons. Ferri-porphyrin also possesses
5 unpaired electrons ; hence all the bonds in the complex are of an electro-
static character. The complex, in which the ferric ion has a coordination
number of 6, can be represented thus :

N

N

OH

N

HjO

• Fe N +

N

H^

I

I

I -'

.1/

HoO

N Fe N

H3O

N

The equilibrium depends upon the pH; addition of HCl will displace it
to the right with the formation of the chloride of ferri-porphyrin which can
be represented as a mixture of the two forms in the following equilibrium :

MACROMOLECULES

115

N

H,0

[sj Fe N

H-O

N

CI

N --

H3O

N

Fe-

ci

N

+ H,0

N

Oxidation of the ferrous ion of a haemochromogen to a ferric ion will
give rise to a parahaematin. One may also obtain a parahaematin by the
combination of a ferri-porphyrin with nitrogen bases; however, the affinity
of ferri-porphyrins for nitrogen bases is much less than that of the ferro-
porphyrins. The parahaematins are slightly paramagnetic; they possess 1
unpaired electron ; the 6 coordination linkages of the complex are covalent
{dhp^) as in the ferricyanide ion, Fe(CN)6, which also has an unpaired
electron. Parahaematin has an octahedral form and can be written so :

NR'

4. Haemoglobin

Combination of globin with a haem gives a haemochromogen of a
particular type : the ferrous ion retains all its 4 unpaired electrons so that
all the linkages in the complex are electrostatic in nature.

Haemoglobin results from the union of protohaem and globin. The
linkage between the globin and the haem is between the ferrous ion and the
two imidazole groups of histidine residues in the globin, the latter replace
the two water molecules in the ferro-porphyrin.

N

; N /

N-

Fe-/'----N

N' .^^
N

In the complex, haemoglobin, oxygen or carbon monoxide can displace
one of the imidazole groups; there is a consequent redistribution of
electrons (bringing 12 electrons to the ferrous ion) with formation of

116

UNITY AND DIVERSITY IN BIOCHEMISTRY

e

•a

c

•a

o
o

g

3

.2

.s

I

o

s

J3

E
2

a

M

c
o

o

d

Iui/u(qoi3oui3EH 3^ = ^ 'uioi— p '(y) 'Soj

Fig. 17 (Reichert and Brown). — Crystals of haemoglobin.
1. guinea pig; 2. grey squirrel; 3. kangaroo; 4. horse.

CH=CH.

-^i.

Vi

■ >

"^'1

(iddy^ffi^pi

u ^ Fox)-Crvstals of chlorocruorin,
Fig. 18 (Roche and tox) ^r^^ ^,.

from

Spirographis Spallan^

anil

MACROMOLECULES

117

6 coordinate covalent bonds {d~sp^) and the appearance of the octahedral
structure. Oxyhaemoglobin and carboxyhaemoglobin are diamagnetic
(same structure as that of the ferrocyanide ion). The detachment of one of
the imidazole groups from the complex and the transformation of the ionic
bond of the other group into a covalent bond, brings about a change in the
pK of the two imidazole groups thus explaining the Haldane effect (change
in the isoelectric point of haemoglobin at the moment of its oxygenation).

I
I

;Fe-'

N globin
Oxyhaemoglobin

N globin
Carboxyhaemoglobin

Haemoglobin can be oxidized to methaemoglobin or ferrihaemoglobin
by oxidizing agents other than oxygen. Methaemoglobin is a parahaematin
of a special type since it contains 5 unpaired electrons, therefore all the
bonds in the complex are electro valent.

Haemoglobin possesses the unique property of complexing reversibly
with molecular oxygen instead of being oxidized by it to ferrihaemoglobin.
It is endowed with this property by the globin which forms with ferro-
porphyrin a complex, which is rather unusual since, unlike other haemo-
chromogens, it is paramagnetic.

The characteristics of the binding of protohaem with a special type of
protein confers on haemoglobin the property used by organisms in many
ways — that of being reversibly oxygenated and deoxygenated without
change in the valency of the iron, which remains in the ferrous state.

Oxyhaemoglobin, like the other haemochromogens, has two absorption
bands in the visible range, in addition to the Soret band which is situated
in the ultra-violet (Fig. 16). Deoxygenation transforms the two-band
spectrum into a one-band spectrum.

5. Chlorocriiorin

Chlorocruorin, which acts as an oxygen carrier in certain types of Anne-
lids, is a derivative of chlorocruorohaem. This latter substance is the haem
of chlorocruoroporphyrin or Spirographis-porphyrin (from the name of the
worm {Spirographis) whose blood is most commonly used as a source of
chlorocruorin) (porphin- 1,3,5, 8-tetramethy 1-2-f ormyl-4- viny 1-6, 7- propi-
onic acid). In fact, it is derived from protoporphyrin by oxidation of the
vinyl group at position 2.

118

UNITY AND DIVERSITY IN BIOCHEMISTRY

log. ^
X

6000 3000 A

Fig. 19 (Roche and Fox) — Visible spectrum of oxychlorocruorin.

lo6|- I.O

0,5

J.

5000 4000 3000 A

Fig. 20 (Roche and Fox) — Ultraviolet spectrum of chlorocruorin.

MACRO MOLECULES

119

6. Cytochromes

These pigments are haemoproteins and are found in all cells which
respire. The form of the haem-protein linkage, in this case, does not allow
oxygen to be involved in the complex, but there is a reversible oxidation
and reduction of iron. The cytochromes show the typical two-banded
absorption spectrum of the haemochromogens when their iron is in the
ferrous state. Since each haemochromogen is characterized by a particular
position of these bands in the visible region, Keilin has been able to detect

(imidazole) N

N (imidazole;

HN— CH — C HN-CH — C

H3C

HC

HaC-

CH

-CH»

CH2 H

CH,

CH,

CH2

]

COOH

COOH

Reduced cytochrome-c

the presence in cells of three cytochromes which he called a, b and c, and
which are characterized by the positions of the absorption bands of their
reduced forms. Cytochrome-^ has been isolated. It has been shown to be
present in all aerobic cells. The protein part of cytochrome-c is rich in
basic amino acids, particularly in lysine.

The haem of cytochrome-c is derived from protophorphyrin, two vinyl
groups being reduced and bound as thioethers to cysteine residues on the
rest of the molecule. The side-chains of the protein are believed to be
attached to the /3 carbon of the vinyl groups, but this has not yet been
completely proved. But, certainly, the a carbon is the point of binding to

120 UNITY AND DIVERSITY IN BIOCHEMISTRY

the sulphur atom. The imidazole nitrogen atoms of the two histidine
residues are firmly attached to the iron, they take the place of the two water
molecules in the haem complex.

Of the a and h cytochromes little is known save that the haem of cyto-
chrome-a is similar to that in chlorocruorin and that the haem of cyto-
chrome-6 is protohaem, Cytochrome-a is not autoxidizable (modification
of the valency of the iron by the action of molecular oxygen) whilst cyto-
chrome-6 is. It has been possible to identify three new haemochromogens
whose spectra are similar to the spectrum of cytochrome -a ; these are cyto-
chromes-^i, -ag and -a^. Cytochromes-ai and -a^ replace cytochrome-a in
certain bacteria where this latter substance is missing. Cytochrome-ag is
identical with cytochrome-oxidase, otherwise known as Warburg's respira-
tory enzyme. In the ferrous state it is autoxidizable, that is, it is oxidized
to the ferric state by molecular oxygen. Cytochrome-«, which is not
autoxidizable, is oxidized by cytochrome-^g (cytochrome-oxidase).

Molecular oxygen oxidizes ferrous cytochrome-flg to the ferric state. An
electron is lost by the iron (which becomes trivalent) and passes to oxygen.
Then the ferricytochrome-ag receives an electron from ferrocytochrome-a
(which becomes ferri-) and is reconverted into ferrocytochrome-^g.

2 Fe++ (cyt. tf ) + 2 Fe+++ (cyt. a^) -^
2 Fe+++ (cyt. a) ^ 1 Fe++ (cyt. a^)
2 Fe++ (cyt. ^g) + O2 -|- 2 H+ -* 2 Fe+-^^- (cyt. a^) + H2O.

7. Hydroper oxidases

In these haemoproteins the iron is in the ferric state and remains in this
state. The reaction catalysed by the hydroperoxidase enzymes is the
following :

AH2 + H2O2 -> A + 2H2O

(A — a phenol, ascorbic acid, etc.)

These enzymes can be divided into two groups : the peroxidases and
the catalases. The peroxidases are principally to be found in plants, but
they have been discovered in milk and in leucocytes. The catalytic action
of peroxidase has been elucidated by B. Chance. He showed that when
H2O2 is added to the peroxidase, a primary addition product is formed
which is green. This enzyme-substrate complex is transformed into a pale
red compound.

MACROMOLECULES

121

Per Fe+++OH + HOOR ^ Per Fe+++OOR + H^O

(brown) (green)

Per Fe+++OOR ^ Per Fe+++OOR

(green) (pale-red)

Per Fe+++OOR + AH2 -> Per Fe+++OH + ROH -f A

Another hydroperoxidase is catalase. This is a particular type of peroxi-
dase which can decompose hydrogen peroxide in the absence of a second
substrate :

2 H0O2 -^ 2 H2O + O2

1

li

I

/

10

k

/

1

b

" \

^

L

\^

300

400 500

m a

600

Fig. 21 (H. Theorell) — Absorption spectrum of crystalline peroxidase.

(b) Chlorophylls and Chlorophyll-proteijis
Chlorophylls-^ and b are extracted from leaves by acetone, along with
other pigments. The addition of an equal volume of petroleum ether and a
small amount of water, to this extract, removes most of the chlorophyll.
From this petroleum ether solution it is possible, by extraction with
aqueous methanol, to remove most of the xanthophyll and a little of the
chlorophyll-6. Extracting with water several times, in this way, one finishes
by precipitating the chlorophyll, which is collected on a filter and its two
constituents separated, by utilizing the fact that chlorophyll-fl is more
soluble in petroleum ether, and chlorophyll-6 is more soluble in methyl alcohol.
The chlorophylls are methylphytol esters of the chlorophyllines, which
are the corresponding acids. These are neutral substances containing
magnesium in a non-ionic form. A hydrolase, called for this reason chloro-
phyllase, splits off from chlorophylls a C-20 (with one double bond) ali-
phatic alcohol which is a diterpene derivative named phytol (see p. 31).

122

UNITY AND DIVERSITY IN BIOCHEMISTRY

When the phytol is separated from a chlorophyll in acetone solution, the
green compound remaining is a chlorophylline.

The formula below shows that the chlorophylls are magnesium com-
plexes of a modified porphin structure, isomeric with protoporphyrin.
Chlorophyll-^ and chlorophyll-6 are present in green plants and the green
algae. Chlorophyll-6 differs from chlorophyll-a by replacement of a methyl
group in position 3 by a formyl group. The structures of chlorophylls c
and d are still not completely known. The brown algae contain chlorophylls
a and c, and the red algae chlorophylls a and d.

Bacteriochlorophyll, which is present in the purple bacteria, differs from
chlorophyll-a in two respects :

H CH, \ H

I c-c=o

HsoQoOOC— CH, I

COOCH,

Phytol

Chlorophyll-a

the replacement of a vinyl group by acetyl, and the reduction of ring II to
a dihydropyrrol structure.

If the magnesium is removed from chlorophyll, a phaeophytin is left, and
if, in addition, the phytol is removed then the remaining fragment is
named a "phaeophorbide", and has the same structure as porphyrin, apart
from the presence of two extra hydrogen atoms and a resulting redistribu-
tion of double bonds.

In the grana of leaves, the chlorophylls are associated with proteins to
form complex macromolecules whose nature is still obscure.

{c) Metalloflavoproteins

Flavin phosphate (FP) or flavin mononucleotide (FMN) (see p. 69)
exist in combination with protein, in a macromolecular form which bears
the name "old yellow enzyme", thus called because others have since been

MACROMOLECULES 123

isolated from yeast and other types of cell. The "old yellow enzyme"
plays a catalytic role in the oxidation of glucose-6-phosphate. Another
macromolecule of the same type is the "new yellow enzyme" in which the
flavin is flavin-adenine dinucleotide (FAD), and which plays a similar
catalytic role to the "old yellow enz}'me", the two being present in the
same cells. Corresponding to the flavoproteins there is a whole series of
metalloflavoproteins resulting from the chelation of copper, iron or
molybdenum.

B. Metalloproteins in which the Metal is Bound Directly to

THE Protein

In proteins, the carboxyl and amino groups are, for the most part,
combined in peptide linkages, so that it is chiefly the polar side chains
which form complexes with metals. If we refer back to Fig. 13, we have a
list of side chains of this type. They are those whose pK is below 10.
Those groups whose pK is above 10 are such strong bases that they cannot
form bonds with metals.

Most protein molecules, then, have many points where complexes may
be formed with metals. Among these metals it is worthwhile to distinguish
those which appear to be coordinated strongly and by many diflferent polar
side-chains : such are mercury, silver, copper and zinc. The alkaline earth
metals, like calcium, seem to be bound primarily by free carboxyl groups,
or, in the phosphoproteins such as casein, by phosphate groups.

The most abundant metals in the biosphere (Na, K, Ca, Mg), which are
bound strongly to proteins, are those whose internal electronic levels are
full whilst the external ones are not. They have the very stable electronic
structure found in the rare gases. They therefore bind to functional groups
by electrostatic attraction whilst the transition elements tend to use their
incompletely filled inner orbits to form covalent bonds.

(a) Proteins Binding Copper

As in the case of the association of a haem and a protein, the nature of
which association controls the diflFerent properties and functions of the
macromolecule, macromolecules of proteins and copper diff^er from each
other, both in their properties and in their functions. Examples of this
type of association are the phenolases and the haemocyanins.

1. Phenolase {phenol oxidase)

This molecule of protein and copper has a double function, as a catalyst
in the o-hydroxylation of phenols and in the dehydrogenation of
o-diphenols.

124

UNITY AND DIVERSITY IN BIOCHEMISTRY
OH OH

OH

►/

v

(O)

Enzyme

W/

V

OH

O

^ (O)

Enzyme

o

V

Similarly, phenolase oxidizes o- and p- polyphenols.
In the course of these reactions, cupric copper of the phenolase is reduced
to cuprous copper which is again oxidized by oxygen.

OH O

2Cu^-+ +

(j

OH

2Cu^

V

o

2H+

V

2 Cu+ + 2 H+ -\- 1/2 O,-^ 2 Cu++ + H.O

There are many uses made of the properties of the cupriprotein phenolase,
both in plants and in animals : respiration, biosynthesis, scelerotization of
cuticles, pigmentation, etc.

2. Haemocyanins

These cuproproteins are oxygen carriers found in certain animals
belonging to the mollusc and arthropod families. They are blue in the
oxygenated state and colourless in the reduced state. The spectrum of
haemocyanin is very similar to that of any copper-protein in which cupric
copper is bound to an -SH group.

The absorption spectra of oxyhaemocyanin in the visible and ultraviolet
regions (Figs. 23 and 24) can be superimposed on the spectra of copper-
serum albumin in which the copper is attached to a sulphhydryl group.

{b) Proteins Binding Iron
Examples of this widely distributed type of compound are the haemery-
thrins, ferritin and transferrin.

1. Haemerythrins

These are large molecules of protein and iron in which the iron, according
to Klotz and Klotz, appears to be partly ferrous and partly ferric. The
haemerythrins which can be oxygenated like the haemoglobins, chloro-
cruorins and the haemocyanins, like these substances, function as oxygen

Fig. 22 (Dhere) — Crystals of the haemocyanin of
spiny lobster.

Fig. 25 (Florkin) — Crystals of haenierythrin from
Phascolosoma elongatum.

MACROMOLECULES

125

700 650

600

550 500

450

mu

Fig. 23 (Redfield) — Spectrum of the haemocyanin of a gastropod {Busycon canalicul atiim
in the visible region. Upper curve : oxyhaemocyanin. Lower curve : reduced haemocyanin)

HEMOCYANIN - - - -

OXYHFMOCYANIN.

Al

r

1

■J

_jj|-^^

'-'

X 600C 5000 4000 3000 2000

Fig. 24 (Roche) — Absorption spectrum of the haemocyanin from the snail.

126

UNITY AND DIVERSITY IN BIOCHEMISTRY

carriers. This use is characteristic of a group of animals, the sipunculids.
The absorption spectra of the haemerythrins are analogous to those of the
haemocyanins. Like the latter, they do not show the absorption character-
istics of haem derivatives that we have noted in the case of the haemoglobins,
cytochromes and hydroperoxidases. Oxyhaemerythrin is wine-red whilst
haemerythrin is colourless. The spectrum of oxyhaemerythrin is like that
of ferriproteins. The iron of haemerythrin, like the copper of haemocyanin,
appears to be attached to an -SH group.

2. Ferritin

Ferritin is a compound of a protein with an iron hydroxide whose formula
is [(FeOOH)8.(FeOP03H2)]. In certain organisms, and particularly in
mammals, it serves as a means of storing iron in organs such as the liver
and spleen.

2

5000

4000

3000

2000

.&

Fig. 26 (Florkin) — Absorption spectrum of the haemerythrin from Sipunculus nudus.

3. Transferrin (siderophilin)

In the blood plasma of mammals there is present a ^-pseudo-globulin
called transferrin or siderophilin, and which at a definite point in its mole-
cule forms an iron complex. This protein, whose molecular weight is in
the region of 90,000, makes up about 3% by weight of the plasma proteins.
The iron is complexed by it in the ferric form and only in the presence of
CO2. When the level of iron in the plasma is at its normal value, which in
man is 129y/100 ml, the transferrin is saturated to the extent of 30% of
the maximum amount of iron which it can carr}\ The same protein can
also fix copper.

MACROMOLECULES 127

V. LIPOPROTEINS

The lipoproteins are macromolecules that are complexes of proteins
with simple or complex lipides. Complexes of steroids and their esters
and carotenoids and their esters with proteins are generally classed under
this heading.

Lipoproteins are very widely distributed in the biosphere. They are
very large and very unstable macromolecules. They are found in all parts
of the cell. Certain authors maintain that the chlorophyll-proteins are
really chlorophyll-lipoproteins. Whatever the truth of this, the study of
lipoproteins has not progressed far.

One of these substances, however, has been the subject of much exhaus-
tive study; this is the lipoprotein of mammalian blood plasma. It is a very
complex macromolecule made up of complex lipides, cholesterol, choles-
terol esters and polypeptide chains.

REFERENCES

GURD, F. R. N. (1954). Chemical Specificity in Biological Interactions. (A collection
of papers read by various authors, the principal theme being the formation
of complexes of proteins with metals and with other molecules.) Academic
Press, New York.

Lemberg, R. and Legge, J. W. (1949). Hematin Compounds and Bile Pigments.
Inter-science, New York.

Wyman, J., Jr. (1948). Heme proteins. Adv. Protein Chem., 4, 407-531.

PART TWO

ENZYMES AND BIOCHEMICAL ENERGETICS

K

CHAPTER I
GENERAL PRINCIPLES OF BIOCHEMICAL ENERGETICS

I. FREE ENERGY

A. Free Energy and Work

The first law of thermodynamics states that the total amount of energy in a
system does not change when the different forms of this energy (chemical,
mechanical, thermal, electrical, etc.) are converted from one to the other.
If a system receives a certain quantity of energy AE, from outside, then the
outside loses the same amount AE' and in all the systems together taking
part in the exchange, the total energy change is zero :

AE= - AE'

Let us consider, for example, a system in the gaseous state. On receiving
from its surroundings an amount of heat Q, it will expand and in doing so
will perform work W, contributing to the energy of the surroundings :

AE=Q-W

But if the system we are considering is unable to perform external work,
as would be the case in a container of constant volume in which an endo-
thermic reaction is occurring whose energy is supplied by the surroundings in
the form of heat, then, we have

AE=Q

The reactions studied in the laboratory are not of the type just described.
They generally take place at atmospheric pressure, that is, with a change of
volume. Let us consider again the above reaction, only this time allowing
the pressure to remain constant, which implies that the volume changes and
work is done on the surroundings. The system will receive the heat Q
from the outside and whilst the endothermic reaction is taking place the
reaction mixture will undergo a change in volume performing positive or
negative work. The change in heat content of the whole system under
consideration is measured by the amount of heat Q, which is divided into
that part which brings about a change in the energy of the reacting system
and another part equal to the work which has been performed.

AH=Q = AE+ PAV
(variation in heat content)

131

132 UNITY AND DIVERSITY IN BIOCHEMISTRY

In fact, if the pressure P remains constant, and the increase in volume is
A V, then the work which has been done is equal to PA V. Since we have
selected an endothermic reaction, AH has a positive value. Its value would
have been negative if an exothermic reaction had been chosen.

At 20° and 1 atmosphere, AH in the case of the combustion of glucose
(CgHiaOe + 60, -> 6H2O + 6CO2) has a value of -673,000 calories per
mole of glucose (heat of combustion), whilst in the case of a mole of palmitic
acid (C16H32O2 + 23O2 -> I6CO2 + I6H2O) its value is -2,380,000
calories.

If we place in a calorimeter, at a temperature of 15° (one calorie = the
amount of heat required to raise the temperature of a gram of water from
14-5° to 15-5°), zinc, mercurous sulphate and water in the molar proportions
in accordance with the following equation :

Zn + HggSO^ + 7H2O -> ZuSO^.THgO + 2Hg

we obtain the products indicated and we can measure a AH value of
—82,000 calories.

However, if we assemble the same components into a battery at 15°
and 1 atmosphere so that it functions perfectly reversibly, the electrical
energy obtained is only equivalent to 66,000 calories. The term free
energy (AF) is given to the maximum work which is obtainable from a
chemical reaction taking place under completely reversible conditions
at constant temperature and pressure. In the case of the reaction
between zinc, mercurous sulphate and water, AH — AF = (—82,000) —
(-66,000) = -16,000 cal/mole.

This represents the energy which is lost (in the form of heat) during the
reaction. Although the first law of thermodynamics states that there is a
definite relation between work and heat, it says nothing about the work
which can be obtained from a given amount of heat. It says only that the
total amount of energy does not change.

The second law of thermodynamics states that there are definite re-
strictions on the transformation of heat into work. The weight which
furnishes a clock with its mechanical energy descends by a spontaneous
process. It is possible, at constant temperature, by the introduction of
external work, to cause the weight to be raised again, but one cannot re-
verse this spontaneous process by supplying heat at constant temperature.
In natural spontaneous processes, a part of the energy liberated is not used
to perform work at constant temperature and pressure. This fraction,
which is not used isothermally, divided by the absolute temperature T, is
the increase in entropy (AS). All spontaneous processes are accompanied
by an increase in entropy. Such a process is the diffusion of a substance iii
solution from a concentrated solution to a less concentrated solution. The
increase in entropy which accompanies such a process can be considered

GENERAL PRINCIPLES OF BIOCHEMICAL ENERGETICS 133

as the result of passing from one state to another with an increase in
disorder, or, if one prefers, a decrease of the ordered state.

To illustrate more clearly the difference between — AH and — AF, that
is, between the heat liberated by a reaction and the maximum possible work
which can be done by that reaction, we may consider molecules as con-
taining two different types of energy. One is an ordered energy : it unites
the atoms to each other by primary or secondary valency bonds. It is this
energy which can do work. The other is of a disordered nature (vibra-
tional, rotational and translational). What primarily interests the bio-
chemist is work, chemical or otherwise, obtainable from a reaction. If the
reaction takes place with liberation of a great deal of useful energy, then
work may be done.

It can be seen that it is AF which we wish to know. But it should be
noted that AF does not depend solely on the nature of the chemical re-
action. It also depends on the concentrations of the reactants, and the
direction of the reaction will also depend on these concentrations. It would
not be practicable to compile tables of AF for all concentrations, so its
value is determined under defined conditions : liquids or solids in the pure
state, gas at a pressure of 1 atmosphere, solutions at 0-lM, temperature
25°. Concentrations thus defined are assigned a value of unity and, from
the AF° value thus defined, it is possible to calculate values of AF for other
conditions.

The use of AF values is limited, as far as the biochemist is concerned.
Pardee compares the information they give with that given to a car-driver
by a map showing contour lines but no roads. The driver, knowing the
power of his engine, can deduce whether he can, or cannot, climb the slope
between two points. No information is given him as to the road to take.

B. Free Energy and the Equilibrium Constant

Under given conditions of temperature, pH, etc., each of the chemical
reactions occurring in the organism is in a stationary state of dynamic
equilibrium (see later). An organism of a given species, given age, and
under specified conditions, has a definite composition. Undoubtedly, in a
species, the compositions of two individuals show differences, but these
differences are much smaller between individuals of one species than be-
tween individuals belonging to different genera, and less still, when
different families, classes or orders, are considered.

A reversible reaction is usually written thus :

A-\- B ^C + D

According to the law of mass action, the velocity of the reaction between
A and B is proportional to the product of the active masses of these two
compounds, and the reaction velocity from left to right is written :

134 UNITY AND DIVERSITY IN BIOCHEMISTRY

., = K,{A) (B)

whilst that from right to left is :

V, = K,{C) (D)

At equilibrium, when v^ = V2, ki{A) (B) is equal to k2{C) (D) and the
equilibrium constant of the reversible reaction is written :

j^ _ (Q jD)
(A) (B)

We can represent a reversible reaction by using small letters for the
number of moles of each reactant.

aA + bB ^cC + dD

The relation between the free energy change and the equilibrium
constant is given by the equation

{CY{DY

AF= -RT\nK+ RT In

{A)^{Bf

K is the equilibrium constant, that is, the ratio between the product
{Cy{D)^ and the product {Ay {By under conditions of thermodynamic
equilibrium. The second term permits AF to be calculated for different
activities. If these latter are equal to unity, the second term disappears
and we have

AF°= -RTlnK

In common logarithms, and replacing the gas constant R by its value
(1-987 cal/degree/mole),

-AF° = 4-575 T log K

C. Free Energy and Electromotive Force

It is known that one can obtain work from certain chemical reactions by
constructing a cell. The maximum work AF can be obtained from a direct
and precise measurement, that of the E.M.F. of the cell. The voltage E is
proportional to the work done for each electron transferred and if it is
known, AF can be calculated from the equation

AF = -nFE

in which n represents the number of electrons transported in the reaction
and F is the Faraday constant.

In oxidation-reduction reactions, as we shall see, the measurement of
E.M.F. provides invaluable information about the free energy of numerous
biochemical reactions.

GENERAL PRINCIPLES OF BIOCHEMICAL ENERGETICS 135

D. Sources of Free Energy

The organisms in Nature obtain their vital energy from two main free
energy sources. One is situated in the biosphere itself, it is the covalent
energy of the organism's food and depends upon the properties of the
external electronic orbits of the constituent atoms.

The other is outside the biosphere : it is nuclear energy coming into it
in the form of light.

Ordinary hydrogen has a nucleus made up of a single proton around
which a single electron revolves.

The atom of ordinary helium has two protons and two neutrons in the
nucleus and two external electrons. If we take the exact masses of a proton
and a neutron, add them together and multiply by two, we obtain a figure
of 4-03304 units. But, if we measure the mass of the helium nucleus, we
shall obtain 4'00279. Between the calculated and the measured value there
is a difference of 0-03025 units, equivalent to an energy value of 28-2 MeV
(millions of electron volts). This is the energy of binding the mass lost in
order to keep together such particles as the protons which, because of their
extremely small size, develop considerable forces of repulsion.

Since this binding energy is 0 in the hydrogen atom and is equal to
28-2 MeV in the helium atom, the transformation of a hydrogen atom into a
helium atom will liberate 28'2 MeV. This is what happens in the centre of
the sun which, because of its considerable diameter (1,392,000 km or 109
times the diameter of the earth), has a resulting gravitational field strong
enough to retain its hydrogen which forms 99% of its weight. The centre
of the sun has a temperature of 20,000,000° C and is under a pressure of
several tens of thousands of atmospheres, so that the kinetic energy of the
hydrogen atoms is sufficient for collisions between them to bring about
nuclear reactions. The sun converts hydrogen atoms into helium atoms by
means of a cyclic process in which carbon acts as a catalyst. It is this
nuclear energy which reaches the biosphere in the form of heat and various
forms of radiation, in particular those which are utilized by organisms
containing chlorophyll.

However, a great many organisms use sources of non-nuclear energy.
This energy is derived from the change in energy level of electrons during
the atomic rearrangements which accompany the changes in structure of
nutrient molecules, and those which accompany oxido-reduction reactions.
These changes in potential are expressed in electron-volts (1 electron-volt
= the kinetic energy acquired by a particle carrying the charge on an
electron when accelerated by a potential of one volt). The commonly used
unit is a million times greater (MeV). The energy corresponding to an
electron-voh is equal to 1-60207 ± 0.00007 X lO-^^ gj-gs. The energy
in calories per mole corresponding to one electron-volt per molecule
= 23-05285 ±3-2 cal/mole).

136 UNITY AND DIVERSITY IN BIOCHEMISTRY

II ENERGY COUPLING

Exergonic reactions are the only reactions which can occur in the bio-
sphere. It is, therefore, necessary to explain by what mechanism cells per-
form numerous biosyntheses where the resulting molecules have an energy
content higher than that of their starting materials.

In the cell, the energy of a chemical bond can be transferred to another
bond by only one possible mechanism, that in which two separate reactions
have one substance common to both. The transfer of energy is accom-
plished by utilizing part of the free energy of an exergonic reaction to bring
about a reaction which, by itself, would be endergonic and would not, there-
fore, otherwise take place.

Consider the following endergonic reaction :
{K^ - 0-01, AF° = +2,470 cal.) Lt equilibrium ^qq^ ^

B\

We are given the value of the equilibrium constant and the reaction will
be at equilibrium when the concentration of ^ is a hundred times that of B,
and it will not take place from left to right except when the concentration
of B is less than one per cent of that oi A. If the concentration of B is
greater than one per cent of that of A, the reaction will take place from right
to left.

Now, consider the exergonic reaction :

(II) B-^C

{K^ = 1000, AF° = -4110 cal.) /'at equilibrium ^

It will take place from left to right until C is a 1000 times more con-
centrated than B.

Now let us suppose that the two reactions occur simultaneously. Re-
action II will continuously remove the product of reaction I, and this
reaction, endergonic when the ratio [5]/[^] is greater than 1/100, will become
exergonic and, consequently, will proceed from left to right, when the
concentration of B becomes sufficiently small for the ratio [5]/[^] to fall
below 1/100. For the combined reaction, AF = (2470 - 4110) = -1370
cal). The overall reaction is therefore exergonic.

The exergonic reaction II causes B to disappear. As B disappears, the
AF of reaction II becomes progressively smaller in absolute value, whilst
the JF of reaction I approaches zero (the reaction becomes less and less
endergonic). When the AF of / becomes negative (because [B] is suffici-

GENERAL PRINCIPLES OF BIOCHEMICAL ENERGETICS 137

ently small), the two reactions proceed in the direction A -> B ^- C uip to
the time when the two AF values become equal to 0, which is the point of
final equilibrium.

III. ENERGY-RICH BONDS

It is to Lipmann (1941) that we owe the classification of the phosphory-
lated compounds which occur in Nature, into two types — those possessing
a bond whose hydrolysis is accompanied by a considerable release of free
energy, and those which yield much less energy. The bonds which are
"energy-rich" from the point of view of the release of free energy by
hydrolysis are, in fact, for the physical-chemist, weak bonds, being rapidly
and easily broken, whilst those bonds which release little free energy on
hydrolysis are stronger and more difficult to hydrolyse. When the different
types of phosphoric esters were listed in the first part of this book (p. 62),
we noted the existence of bonds of the acid anhydride type, resulting from
the union of two molecules of acid with elimination of a molecule of water.
These bonds are hydrolysed with the release of large amounts of energy
as can be seen, for example, when acetic anhydride is mixed with water :
the reaction takes place with a considerable increase in temperature.

The most important type of acid anhydride in biochemistry is that
between two molecules of phosphoric acid, as exemplified by the two ter-
minal bonds of ATP (p. 67), but the pyrophosphate bond is not the only
type of energy-rich bond as can be seen from Table XI. Mixed anhydrides
of carboxylic and phosphoric acids (acyl-phosphates), phosphondated
enol groups (enolphosphates), phosphorylated guanidine groups (guanidine
phosphates), compounds of sulphhydryl groups with phosphoric or car-
boxylic acids (thioesters or thiophosphates), all these types of compound
are energy-rich.

How does one explain this release of large amounts of free energy when
an energy-rich bond is hydrolysed ? As far as the pyrophosphate linkage is
concerned, one of the reasons is that in pyrophosphate the number of
resonating structures is much smaller than in inorganic phosphate. Also,
in the pyrophosphate molecule there are several Hke charges close to each
other and their reciprocal repulsion is balanced by a certain amount of
energy which is set free on hydrolysis. Moreover, the neutralization of the
acid groups liberated on hydrolysis is also productive of energy. Con-
siderations of a like character can explain the liberation of energy which
accompanies hydrolysis of the acyl-phosphate and guanidine-phosphate
bonds, but they cannot explain this in the case of the phosphoenolpyruvate
bond. In this case, one of the sources of the energy is the transformation
of the enol form of pyruvic acid into the keto form.

The formation of glycogen from glucose is an example of an apparently
endergonic reaction which is, in fact, made exergonic by the mediation of a

138 UNITY AND DIVERSITY IN BIOCHEMISTRY

molecule having an energy-rich bond, ATP, which acts by phosphorylating
one of the reactants : —

glucose + (glycogen)^ -^ (glycogen)n+i + HgO AF° = +5,000 cal

The reaction can be split up into the following stages :

glucose + ATP -^ G-6-P + ADP + H+ AF° = -8,000 cal

G-6-P ^ G-l-P AF° = +1,800 cal

G-l-P + (glycogen)^ -> (glycogen)„+i + H3PO4 AF° = + 200 cal

The sum of these reactions leads to the following :
glucose + (glycogen)„ + ATP -> (glycogen)„+i

+ ADP + H3PO4 + H+ AF° = -6,000 cal

IV. THE PHOSPHATE CYCLE

Research carried out during recent years has revealed that the energy
stored in food which is broken down during cellular metabolism is gradu-
ally liberated in "packets" of energy which can be stored and utilized at a
later date. The direct oxidation of glucose by oxygen :

6O2 + CeHiaOe -> 6CO2 + 6H2O AF° = -686,000 cal

will release a considerable amount of energy in the form of heat, that is to
say, in a non-utilizable form. But, as Pardee has remarked, one does not
start a car by putting a match to the petrol tank.

The energy economy of living organisms rests on the fact that anaerobic
and aerobic metabolism can give rise to energy-rich bonds, which can be
stored as energy-rich bonds of ATP and this energy can be used by the cell
to do various forms of work including the chemical work of biosynthesis.

V. BIOLOGICAL OXIDO-REDUCTIONS AND THE GENERATION

OF ENERGY-RICH BONDS

Energy-rich bonds are generated by the conduction of certain definite
electrons along definite channels. During this passage of electrons their
energy level is lowered and energy-rich bonds are formed at their expense.
So an organism is an electro-chemical machine whose structure is that of
a special type of electronic conductor.

A. Oxido-Reduction Potentials

Consider a solution of ferric chloride. In this solution there are Fe+'*"+
ions, CI- ions and water molecules. The solution is stable, however it is
capable of accepting electrons with the conversion of Fe+++ to Fe++ : we
can say that it has a certain (negative) electron pressure. We can measure
this pressure, provided that we have a reference system which we can place
together with the solution in an electric circuit. Let us place in the ferric

GENERAL PRINCIPLES OF BIOCHEMICAL ENERGETICS 139

Table XI
Free energy liberated in the hydrolysis of various bonds

Bonds

Oside linkage

Peptide linkage

O

Ester linkage R — C-

R-O
H

O-R'

-PO3H2
OR

c ^

no,

R_CJ-NH-R'

Kilocals jmole
(negative values)

2.0 - 4.0

4.8

3.0

Pyrophosphate

Guanidinephosphate

Enolphosphate

Acylphosphate

Thioester

Thiophosphate

O

II

R— P-O

I

O

II

^P— R'

I

OH OH

NH ,

" I

R-NH-C-NH-fPOaH^

R=:C-0~P0aH2
I

o

II I

R-C-O-l-POaHa

O

R — C4SR

R— S-

12.0

PO3H,

14.0

16.0

16 0

16.0

ca. 16.0

140 UNITY AND DIVERSITY IN BIOCHEMISTRY

chloride solution an electrode made of a noble metal (platinum, for
example) at whose surface no chemical reaction occurs. By means of an
agar bridge, we can connect the solution with another solution capable of
giving or receiving electrons. In this solution we will also place a platinum
electrode and we will join the two electrodes by a wire together with a
potentiometer in the circuit. If the electron pressures of the two solutions
are different there will be an electron flow which will be revealed by the
passage of an electric current. If we know the electron pressure of the
reference solution, and measure the potential difference and direction of
flow of the current, then we have a basis on which to calculate the electron
pressure of the first solution. As a reference solution we may take what is
commonly known as the normal hydrogen electrode, that is, a normal solu-
tion of hydrochloric acid saturated with hydrogen gas. The e.m.f. of the
system is measured with a potentiometer. The electron pressure (which
depends upon the ratio between the concentrations of the oxidized and re-
duced substance) is called the oxidation-reduction (or redox) potential (E).
The relation between E and the concentrations of the oxidized substance
and the reduced substance is given by the equation :

RT (oxidized form)
nF (reduced form)
in which,

R is the gas constant;

T is the absolute temperature ;

n is the number of electrons involved when the substance passes from

the oxidized to the reduced state (for Fe+++ <^ Fe++, « = 1);
Eo is a special constant for each particular system.
Furthermore, In signifies log to the base e and F is the Faraday {F =
96,500 J, and since 1 cal = 4-18 ], F= 23,098 cal).

If the concentration of the oxidized form (Ox) is equal to the concen-
tration of the reduced form (Red),

(Ox)
{Ox) = {Red) and In -j^^ = 0.

RT

£:=£„+ -J- XO=Eo.

To measure Eo, it is only necessary to measure E when {Ox) = {Red).
Eo is termed the standard oxidation-reduction potential, it is the potential
which is measured against the hydrogen electrode at the pH of a normal
solution of HCl (pH = 0, since log 1 = 0). It has become customary to
state the potential at, or around, pH 7-0 and this standard potential is
designated by the symbol Eo'.

GENERAL PRINCIPLES OF BIOCHEMICAL ENERGETICS

141

In Table XII is listed a series of Eo values for a number of systems
important biochemically. If system A is characterized by a higher (or
less negative) value of E than system 5, then B can serve as a reducing
system for system A, and A as an oxidizing system for B.

We have seen (p. 134) that there is a definite relation between the change
in free energy AF and electromotive force E, and that by means of the
following equation it is possible to calculate the electrical work accompany-
ing the formation of a mole of reaction-product.

AF

■nFE

Let us now consider, inside a cell, the oxidation (or dehydrogenation)
of lactate to pyruvate, the proton and the electron of the hydrogen, or the
electron alone, being conducted through several intermediates as far as
cytochrome-c. Let us also suppose that the pyruvate and lactate are
present at equal concentrations and that the cytochrome-c is 50% reduced
and 50% oxidized. The number of intermediates does not matter. E' o
for the lactate-pyruvate system = 0-180 at pH 7-0; at the same pH, for
the system cytochrome-c (Ox)/cytochrome-c (Red), E' o = 0-262. Since
both systems are 50% reduced, E = E' o for both,

and zl5 = 0-442 volts
whence -AF =2x 96,500 x 0-442 = 85,500 J = 20,500 cal.

So that the energy liberated by the oxidation of a mole of lactate by a
mole of cytochrome-c under the specified conditions is 20,500 calories.
Johnson calculates that this amount of energy would be sufficient to keep a
100 W lamp alight for 14 min.

Table XII
Some oxidation-reduction potentials

System

Temp.

pH

Eol

(volts)

a-ketoglutarate-succinate

7-0

-0-600

Acetaldehyde-acetate

7-0

-0-468

H+-hydrogen

All. temps.

0-0

-0-060

Coenzyme I

30

7-0

-0-282

Lactate -pyruvate

35

7-01

-0.180

Malate-oxaloacetate

38

7-0

-0-102

Succinate-maleate

37

7-0

-0-094

Cytochrome-6

20

7-4

-0-04

Succinate-fumarate

7-0

0-00

Cytochrome-c

7-0

+ 0-262

Cytochrome-a

20

7-4

+ 0-29

142 UNITY AND DIVERSITY IN BIOCHEMISTRY

B. Formation of Energy-rich Bonds during Oxidation-reduction

Reactions

Although the passage of electrons, either from one point to another in
the same molecule, or by a conductor made up of a series of systems of
higher and higher oxidation potential, is accompanied by a change in free
energy, these free energies, like that resulting from the direct oxidation of
glucose, are not in a form which can be used by the cell. These free ener-
gies will be degraded into heat and lost to the cell unless some mechanism
exists to store them in the form of energy-rich bonds of ATP, the universal
source of cellular work.

It is, therefore, important now to consider this mechanism — the coupling
of electron transfer and phosphorylation. There are two aspects to be
considered : phosphorylation at substrate level and phosphorylation during
the transfer of electrons through a series of intermediates.

(a) Phosphorylation at Substrate Level

An example of this type of genesis of pyrophosphate linkages is furnished
by the oxidation of phosphoglyceraldehyde to 1,3-diphosphoglyceric acid,
an oxidation which occurs during glycolysis.

Phosphoglyceraldehyde dehydrogenase (PGAD) is the enzyme catalys-
ing the oxidation (anaerobic) of phosphoglyceraldehyde which takes place
with an internal redistribution of electrons and the accumulation of 16,000
calories in the acyl-phosphate bond. The enzyme, whose coenzyme is

C C

^H

^ 0-P0(0H)2

CHOH -t-DPN^ +H3PO4 ^ CHOH +DPNH

CH20PO(OH)2 CH,OPO(OH)2

Phosphoglyceryldehyde 1,3-diphosphoglyceric acid

DPN, has been crystallized. It is poor in cysteine but rich in basic amino
acids. Near to an — SH group on the surface of the enzyme protein
molecule there is attached a molecule of the coenzyme DPN+. An addition
complex is formed between the — SH grouping and the — N = C — of
DPN+. When phosphoglyceraldehyde is added, this bond is broken and
an energy rich thioester is formed and at the same time the DPN+ is
reduced to DPNH. A phosphate residue which is also attached to the
protein molecule takes the place of the thiol group and the energy of the

GENERAL PRINCIPLES OF BIOCHEMICAL ENERGETICS

143

thioester bond is transferred to the acyl-phosphate bond. 1,3-diphos-
phoglyceric acid subsequently detaches itself from the protein molecule.

.O

\

— H,0

OH

CHOPO(OH),

CH.OH

2-phosphoglyceric acid

OH

CO-PO(OH);

CH,

phosphoenolpyruvic acid

— »

o

OH

^

O

OH

CO--PO(OH), +ADP ^ COH -f- ATP

CH2

phosphoenolpyruvic acid

CH,

pyruvic acid (enolic form)

^

-^

V

o

OH

c-=o

CH3

pyruvic acid (ketonic form)

This reaction can only continue till all the substrate is used up, if the
reaction product, which is 1,3-diphosphoglyceric acid, is removed
from the solution ; in the cell, this acid then reacts with ADP, converting
it into ATP; the energy-rich anhydride bond then disappears

144 UNITY AND DIVERSITY IN BIOCHEMISTRY

from the 1,3-diphosphoglyceric acid and reappears in ATP.
1,3-diphosphoglyceric acid + ADP:<^ 3-phosphoglycericid + ATP.

This transfer of energy of the energy-rich acyl-phosphate bond to the
energy-rich pyrophosphate bond is catalysed by a highly specific enzyme,
3-phosphoglyceric phosphokinase ; this enzyme, also, has been crystallized.

An example of the transformation of an ordinary phosphate ester into an
energy-rich phosphate is provided by another reaction of the glycolysis
chain : the conversion of 2-phosphoglyceric acid into phosphoenolpyruvic
acid followed by a transphosphorylation. The reaction (a dehydration),
catalysed by enolase, results in the redistribution of the internal energy of
the molecule with concentration of around 16,000 calories in the enol-
phosphate bond.

The rapid transfer of this energy-rich phosphate to ADP with formation
of ATP is brought about by pyruvic phosphokinase.

(b) Phosphorylation in the Respiratory Chain

As we have just seen, the formation of ~P bonds is coupled with
oxidation-reductions taking place at substrate level. Others may be formed
in a way which depends on the energy liberated by electrons removed from
the substrate as they pass along a series of carriers of increasing potential
until finally oxygen is reached. Acetyl-CoA resulting from a number of
metabolic reactions (see Part 3) is oxidized by a common pathway in the
presence of oxygen. Here, unlike the previously cited examples taken from
anaerobic glycolysis, the phosphorylations which yield pyrophosphate
bonds are not coupled to oxidation-reductions at substrate level. This
concept is based on a number of experimental findings. The quantitative
study of this process was initiated by Belitzer in 1939. As we shall see,
during the respiratory cycle which transforms substrates into COg and
HgO, a series of dehydrogenations occur. If each of these dehydrogen-
ations was accompanied by a phosphorylation of the substrate, then for
each of these a molecule of phosphate would be removed from the solution
and an atom of oxygen would be consumed (P/0 would be equal to 1).
Now, Belitzer calculated the ratio P/0 and obtained values greater than
unity. This expresses the fact that for each pair of electrons (or of electrons
accompanied by protons) transferred to oxygen several phosphorylations
occur. Theoretically there is nothing very astonishing about this since
when we defined oxidation-reduction potentials we showed that when
lactate is dehydrogenated to pyruvate, the transfer of electrons or hydrogen
atoms (electron + proton) to oxygen liberates much more energy than is
required for the formation of an energy-rich pyrophosphate bond.

Belitzer put forward the following hypothesis, which has been confirmed
by numerous observations : the reaction producing the energy which is
coupled to phosphorylation is the oxidation of the carrier, which was re-

GENERAL PRINCIPLES OF BIOCHEMICAL ENERGETICS

145

duced directly by the electrons of the substrate by a second carrier of
higher oxidation-reduction potential ; the carrier of the highest oxidation-
reduction potential is, of course, oxygen itself.

This coupling of phosphorylations to oxidation-reduction during respi-
ration makes up the dynamo which transforms the energy of electron
transfer into energy-rich bonds of ATP. Without this coupling this energy
would be lost in the form of heat, and, in fact, a part of it is lost in this way.
We shall leave a detailed discussion of this aspect of energy until we come
to study the particular reactions involved.

VI. THE CELLULAR DYNAMO

The preceding has given us some idea of the lay-out of the cellular
machine and the function of its "dynamo" (Fig. 27). Details will be studied
later. This scheme is of general application for the provision of energy to
the living cell from the packets of chemical energy in the form of cellular
nutrients. However, there is another way of obtaining energy — by utilizing
the energy of the sun to perform biosynthesis. This dynamo functions by
coverting electromagnetic energy into chemical energy, but at the present
time its mode of operation is not so well understood as the chemical energy
dynamo described above.

However, even if not generally accepted, the scheme proposed by Calvin
is of interest. The first stage of photosynthesis, is the conversion of water
into a reducing substance and half a molecule of oxygen :

hv

Heat

HP

Biosynthesis

2[H] + JO,

Mechanical
Work

Active Bioluminescence
Transport

/_

oxido-reduction

Fig. 27 — General lay-out of the cellular dynamo (modified from Lipmann)

146

UNITY AND DIVERSITY IN BIOCHEMISTRY

In Calvin's view, in the organized and oriented system containing chloro-
phyll, absorption of light liberates an electron. As depicted in Fig. 28,
water is on one side of the photobattery and the disulphide form of thioctic
acid on the other. Electrons move towards the thioctic acid layer and the
sites which are left empty are immediately filled up by electrons coming

CO2 COj

H

o— H

o— H

I

H

SH SH

TPNH

0 •

o +

+

1 o

o

o +

■"0 •

2H00

H

I
O— H

O— H
I
H

1^
2H-

[H2O,]

\H20

Fig. 28 (after Calvin) — The role of thioctic acid in the first stage of photosynthesis.

from the water molecules. In particular, this photobattery produces a
high concentration of TPNH.

For the moment, the scheme in Fig. 28 is sufficient to illustrate the
fact that the energy-transformer at the primary stage of photosynthesis is
different from the chemical type of transformer. Later, we shall return to
the question of photosynthesis.

VII. THE PYROPHOSPHATE BOND AND CELLULAR WORK

The pyrophosphate bonds of ATP are the coins which pay for the
performance of cellular work, and there are as many examples of their use
in this way as there are types of dynamic biochemical reaction.

A very common type of cellular work is the transport of a molecule
against the concentration gradient, for example, the transport of glucose
from a region where its concentration is low, through a membrane, to a
region where the concentration of glucose is higher. Such a case is
represented in Fig. 29. On the left-hand side of the membrane, corre-
sponding to the low concentration of glucose, the glucose is phosphorylated
in the presence of hexokinase at the expense of a molecule of ATP and
G-6-P is formed. The presence of this molecule, which diffuses freely in
the thickness of the membrane, does not prevent the diffusion of glucose to
continue through the left-hand face. On the right-hand side, this ester is
hydrolysed in the presence of a phosphatase, regenerating glucose and

GENERAL PRINCIPLES OF BIOCHEMICAL ENERGETICS

147

liberating inorganic phosphate ; the concentration of glucose on this side of
the membrane is above that existing on the right of the membrane ; so the
glucose diffuses to the right of the membrane. The membrane is made up
of cells and its thickness is the same as these cells. Thus, the free energy of
a pyrophosphate bond is used for each molecule of glucose transported,
and it is converted into work done against the concentration gradient.

Low

concentration

ATP ADP

Hexokinase

G-

Membrane

;

Phosphatase

- G

High
concentration

Fig. 29 (Cantarow and Schepartz)— Transfer of a glucose molecule against the
concentration gradient at the price of an ATP pyrophosphate bond.

VIII. CHEMICAL EQUILIBRIUM AND THE STATIONARY STATE

Let us recall the definition of a reversible reaction : it is a reaction in
which the reacting substances are not completely used up in forming the
products of the reaction. If, for example, we mix equimolecular amounts
of ethyl alcohol and acetic acid, water and an ester will be formed according
to the equation

C0H5OH + CH3COOH = CH3COOC2H5 + H2O

Whatever the duration of the reaction, at equilibrium a third of the ethyl
alcohol and a third of the acid will remain unchanged, in the presence of
the reaction products from the other two-thirds.

If, on the other hand, we mix equimolecular amounts of the ester and
water, ethyl alcohol and acetic acid will be formed

CHgCOOCoHg + H2O = C2H5OH + CH3COOH

But two-thirds of the reactants will remain unchanged in the presence
of the products from the remaining third.
So, one can write

C2H5OH + CH3COOH ^ CH3COOC2H5 + H2O

The reaction is reversible. Whether the reaction begins from the left or
the right it will reach equilibrium when there are twice as many ester
molecules as there are alcohol molecules.

148 UNITY AND DIVERSITY IN BIOCHEMISTRY

In a reversible reaction it is the difference in the reaction velocities in
opposing directions which regulates the position of equilibrium. In effect,
all reactions are reversible, but if the velocity from left to right is very great
and the velocity from right to left very small, then the reaction may be
considered as irreversible.

However, even though the reaction velocity is appreciable in either
direction, one of the reaction products may be constantly removed, and in
such a case a reversible reaction can go to completion in one direction. For
example, if we react iron and water vapour in a closed vessel we shall have
the reversible reaction :

3Fe + 4H2O ^ FegOi + W^

Now, if we pass the water vapour over the iron, the hydrogen will be
swept away and the reaction will go to completion from left to right :

3Fe + 4H2O ^ Fe304 + 4H2

If, on the other hand, we pass a stream of hydrogen over the heated iron
oxide, it will be the reaction from right to left which will go to completion,
the current of gas sweeping away the water vapour :

FegO^ + 4H2 -> 3Fe + 4H2O

The equilibrium of a reversible reaction is the resultant of velocities in
the two directions. Temperature influences these velocities but it does not
change them relative to each other, nor consequently the position of
equilibrium. The presence of a catalyst will influence the velocities but
likewise will not alter the point of equilibrium. It is the concentrations of
the substances present which control the direction of a reversible reaction,
as stated by the law of mass action.

In living organisms, it happens frequently that the products of a rever-
sible reaction are used in another reaction, or that they are removed from
the site of their formation. Inside the cell, a reversible reaction often goes
to completion in one direction and becomes, in effect, an irreversible
reaction.

A closed system can only do work at the cost of an increase in entropy.
And irreversible reactions can only take place in one direction, that of an
increase in entropy. The reactions will continue until equilibrium is
established.

The situation is quite different in an open system or in an assembly of
open systems, as is the case for a cell or an organism. In such an open
system, the organism takes complex organic molecules from the environ-
ment, liberates free energy from these molecules, and rejects the products
of the reaction. It can maintain its entropy constant and even decrease it
so that its constituents become more ordered. It has been described as

GENERAL PRINCIPLES OF BIOCHEMICAL ENERGETICS 149

being nourished by negative entropy (Schrodinger). There is nothing here
which contradicts the second law of thermodynamics, for in the total
system made up of the organism and its surroundings the entropy increases.
An organism is made up of one or more cells ; each cell is itself an assembly
of open systems which receive free energy and dispose of it.

As Prigogine (1947) has shown, open systems have some very remarkable
properties.

(1) They tend towards a "stationary state" corresponding to a state of
minimum entropy compatible with the conditions of the system.

(2) The entropy of the system can decrease when the "stationary state"
is established.

(3) If one of the components of the system is modified, the system
changes in an opposing direction, revealing a capacity for self-regulation.

It can be seen that the statiojiary state of "constrained disequilibrium",
which is manifest in organisms, with its continual introduction and removal
of materials, is quite different from the equilibrium of a reversible reaction.

Let us take, for example, the case of a monomolecular reversible reaction

k

A ^B

k'

According to the law of mass action, we may write :

(^) k ^ ^ ...^ .

--J- = -,= K (equilibrmm constant)

[A) k

When equilibrium is reached, the rates of conversion of A into B, and B
into A, are equal.

If B is constantly removed to a pool Z and if A is constantly replaced
from a source of supply S, the flux will be defined as follows :

k« k kz

S< >A ^B< >Z

k'

ks and kz are the diffusion constants (or "permeabilities"). It can be seen
that to define the stationary state we must consider not only the size of the
source and the pool but also the equilibrium constant of the reaction itself.
In order for the stationary state to be maintained, S and Z must remain
constant regardless of subtractions or additions.

There are many cases in organisms where the equilibrium of a reversible
reaction has been upset in one direction or the other. For example, in
vertebrates when oxygen is transported, the reversible reaction

Hb + 0.2 ^ HbO,

takes place from left to right when the blood passes through the lungs and
from right to left when the blood reaches the tissues.

150 UNITY AND DIVERSITY IN BIOCHEMISTRY

But this is not the case in the reactions of intermediary metaboHsm.
When these are under consideration, one must always bear in mind the
complex stationary states of open systems, in which the equilibrium is not
limited to the consideration of only one reaction and is not controlled solely
by the equilibrium constant of a reversible reaction.

REFERENCES

Bladergroen, W. (1955). Einfuhrung in die Energetik und Kinetik biologischer

Vorgdnge. Wepf, Basel.
Johnson, M. J. (1949). Oxidation-reduction potentials. Respiratory Enzymes,

edited by Lardy, H. A., Burgess, Minneapolis, revised edition, 58-70.
Johnson, M. J. (1 949) Energy relations in metabolic reactions. Respiratory Enzymes,

edited by Lardy, H. A, Burgess, Minneapolis, revised edition. 255-263.
Pardee, A. P. (1954). Free energy and metabolism. Chemical Pathzvays of

Metabolism, edited by Greenberg, D. E., vol. I, Academic Press,

New York, 1-25.
Prigogine, L (1955). Introduction to Thermodynamics of Irreversible Processes.

Thomas, Springfield.

CHAPTER II
ENZYMES

I. DEFINITION

In order for a reaction to take place spontaneously, we have seen that it
must have a negative AF. But this condition alone is not sufficient to say
that the reaction will take place. The petrol in the tank of a car has a very
negative AF for the oxidation reaction, but it remains stable in air. Simi-
larly, food in a grocer's shop, in contact with air, is also stable, although this
food is destined to provide much energy in the oxidations it will undergo,
in the presence of oxygen, in the human body. In order to react, most
molecules have to be activated. The act of bringing a lighted match to the
surface of the petrol accomplishes the activation.

Conforming to the concept of activation introduced by Arrhenius, the
energy content of molecules is not constant but is continually changing.
Certain molecules, the activated molecules, have an energy higher than the
other molecules, and only they are capable of entering into a reaction. In a
solution of sucrose, for example, the number of activated molecules is
extrem^ely small but if the temperature is increased by 10° their number is
increased two or three times. These activated molecules travel faster and
are more labile.

Uncatalysed reaction

Catalysed reaction

Reactants

Reaction products

Fig. 30

When the molecules have absorbed a certain amount of energy {E in
Fig. 30), reaction takes place between the reactants and the reaction pro-
ducts are formed. Nevertheless, the need for activation can be reduced, to

151

152 UNITY AND DIVERSITY IN BIOCHEMISTRY

varying degrees, by the presence of catalysts, which, in effect, lower the
activation-energy barrier. In Nature, reactions do not take place at high
temperatures, and most of them only occur because of the presence of the
organic catalysts which we call enzymes.

As we saw in the preceding chapter, one of the characteristics of the
biochemical machine is a constant opposition to the establishment of
thermodynamic equilibrium.

A cell, even if after a given period we do not observe any change in its
composition, is not in a state of thermodynamic equilibrium, but is in a
stationary state of flux in which the velocities of synthesis and breakdown,
for example, are equilibrated. Such an equilibrium is the result of the
control of the velocities in question. The control of these velocities is the
work of the very many specific catalysts which each cell contains.

II. ENZYMES AND ACTIVATORS

It was in 1926 that an enzyme, urease, was crystallized for the first time
by Sumner. Since then, many other enzymes have been crystallized and
the list is constantly increasing. All these purified enzymes are proteins.
However, as long ago as 1897, Gabriel Bertrand introduced the name
"coenzyme" or "coferment" for those metal ions whose presence was
indispensable for the action of certain enzymes.

It soon became evident that certain enzymes were heteroproteins in
nature, that is that they contained a prosthetic group firmly attached to the
protein. For example the haem in catalase. Also there are a number of
cofactors in the absence of which biocatalysis does not occur. At the present
time the term coenzymes refers to these small organic molecules which are
indispensable for the performance of biocatalysis but which are not in-
cluded in the enzyme molecule in the state in which it is isolated from the
cell. Hence enzymes exist which are inactive in the absence of the co-
enzyme.

In addition, when an ion, whether attached firmly or not to the protein, is
indispensable for its biocatalytic action, it is called an activator. The study
of the mechanism of enzyme action, of coenzymes and activators, is in full
swing at the present time. We shall, therefore, content ourselves for the
time being with the above terminology which covers extremely diverse
mechanisms. Some examples of these general biochemical mechanisms will
be taken and described in the pages which follow.

III. CLASSIFICATION OF ENZYMES
A. Hydrolases

These catalyse reactions of the type : AB + HgO -^ AGH + HB.
These enzymes can be considered as catalysing the transfer of a trans-
ferable group with water playing the role of specific acceptor. They can

ENZYMES

153

be divided into several types according to the nature of the donor system
and the transferable group. The general type of transfer reaction and the
particular case where water is the acceptor can be represented by the
following two schemes of reaction :

R_X+HE ^ R— E+XH R-X+HE ^ R— E+XH

f ' \

YH H OH

R— Y+HE R— OH+HE

in which R — X is the substrate (donor system), R is the transferable group,
Y is the acceptor system and E is the enzyme.

Hydrolases catalyse the transfer of transferable groups to water. If
another acceptor is present, in sufficient quantities and in the presence of
the specific enzyme for the activation and transfer of the group to this
other acceptor, then this reaction will compete with the hydrolysis.

The donor system varies according to the various types of hydrolases
and the transferable group may be attached by a peptide bond, oside bond,
ester bond, amine linkage, amide bond or an amidine linkage.

In all living beings, as a rule, seven types of hydrolases are recognized.

O

O

(a) Peptidases

RC— NHR' + H.,0 ^ RC-OH + NH.R

(b) Carbohydrases R' — O— R' + HoO ^ R'OH + R'OH

(R^ and R^ are sugar residues or osides, they can be identical.)
O O

II II

(c) Esterases R'— C— OR' + H,0 ^ R^C— OH + R'OH

(d) Phosphatases R— O— PO3H2 + H2O ^ ROH + H3PO4

(e) Deaminases

R— NH2 + H2O ^ ROH + NH3
O O

(f) Deamidases R— C— NH^ + H2O ^ R— C— OH + NH3

NH

(g) Deamidi- II

nases R— NH— C— NHo + H2O ^ R— NHo + H2NCONH2

154 UNITY AND DIVERSITY IN BIOCHEMISTRY

When the transferase systems (transphosphorylases, transglycosidases,
transpeptidases, transmethylases, transacylases, etc.) which function inside
the cell, cease to act and compete with the transfer to water, the hydrolases
take over and cause a certain amount of hydrolysis of the cellular contents.

(a) Peptidases

All cells contain peptidases, and in many specialized cases they secrete
enzymes of this type to the exterior ; this is the case, for example, with many
bacteria, and with certain cells of the digestive tract of animals, etc. There
are many special aspects of this subject which will not be discussed here.

The peptidases are divided into endopeptidases and exopeptidases.
The endopeptidases (formerly proteinases) are able to attack all the peptide
bonds in a molecule, even those which are some distance from terminal
groups, whilst the exopeptidases (formerly peptidases) can only hydrolyse
peptide bonds at the ends of the chain. Among the exopeptidases, some
remove terminal residues having a free carboxyl group (carboxypeptidases)
whilst others remove those where the amino group is free (aminopepti-
dases).

Still little is known about the system of intracellular peptidases. Some
studies have been made of the so-called cathepsin, which is the intracellular
peptidase system of mammalian kidney and spleen. The studies of Berg-
mann and his collaborators have revealed that the system contains endo-
peptidases, carboxypeptidases and aminopeptidases.

[b) Carbohydrases

Like the peptidases, these enzymes are universally found in the bio-
sphere. They are considered under two headings, glycosidases which
hydrolyse di- and trisaccharides and glycosides, and polysaccharases
which hydrolyse macromolecules such as starch or cellulose.

The glycosides possessing a free reducing group can exist in the a form
or the j8 form. One can also characterize the reducing group involved in
the glycoside linkage by referring to a-glycosides and to ^-glycosides. Mal-
tose, for example, is an a-glucoside, lactose a j8-galactoside, and sucrose,
at the same time, is both an a-glucoside and a ^-fructoside.

There are a certain number of glycosidases which are specific for a given
linkage regardless of the molecule which contains it : a-glucosidase
(formerly maltase), ^-glucosidase (formerly emulsin, cellobiase, gentio-
biase), a-galactosidase, /S-galactosidase, j8-fructosidase (formerly invertase,
saccharase, etc.), a-mannosidase. In addition, there are a number of gly-
cosidases which are specific for a given compound (for example, trehalase
which acts only on trehalose and not on other a-glucoside linkages).

The glycosidases are without action on polysaccharide macromolecules,
although starch and glycogen, for example, consist of chains of glucose

ENZYMES 155

molecules joined together by a-glucoside linkages. The hydrolysis of
starch only takes place in the presence of amylases, ^-amylase attacks the
long chains of amylose and hydrolyses it completely. With amylopectin
(or with glycogen), ^-amylase acts on the outer chains but its action is
stopped at the point of branching. When acting on amylopectin, j8-
amylase hydrolyses it to maltose to the extent of 65% and the residue is
attacked by a-amylase.

Whilst a-amylase appears to be very widely distributed in living beings,
up to the present /3-amylase has not been detected in animals.

A whole series of other polyases exists and they are more or less widely
distributed in the biosphere : cellulases, dextranases, lichenases, inulinases,
pectin-polygalacturonidases, mucopolysaccharases, lysozymes, hyaluroni-
dases, etc.

(c) Esterases

These enzymes hydrolyse esters of organic acids and alcohols. They are
widely distributed but, in general, their specificity is of a low order.

It is possible to divide them into the true esterases (catalysing the hydro-
lysis of all esters of monocarboxylic acids and monohydric alcohols) and the
lipases (catalysing the hydrolysis of esters of fatty acids and glycerol).
However it is not always easy to make this distinction because of the low
specificity of these enzymes.

In certain regions of the biosphere are localized esterases possessing a
more marked specificity : chlorophyllase, pectin-esterases, cholinesterases, etc.

Besides the esterases for esters of organic acids, there are esterases for
the esters of inorganic acids. A widely distributed group is that of the
sulphatases or esterases for esters of sulphuric acid.

(d) Phosphatases

Phosphatases are present throughout the biosphere. They can be divided
into four groups: —

1 . Phosphomonoesterases

O
R-O— P— OH + H2O ^ ROH + H3PO4

OH

These are of low specificity as far as the alcohol radical R is concerned.
In this category are included alkaline phosphatase, acid phosphatase,
acetylphosphatase, hexosediphosphatase, etc.

156 UNITY AND DIVERSITY IN BIOCHEMISTRY

2. Phosphodiesterases

O Q

II II

R^— O— P— OR' + H.O ^ R^— O— P— OH + K'OH

I I

OH OH

Likewise their specificity with respect to R^ and R^ is low. In this group
are such universally distributed enzymes as ribonuclease and desoxy-
ribonuclease.

3. Pyrophosphatases

OH OH OH OH

0=P— O— P=0 + H2O -^ 0==P— OH + 0=P— OH

II II

OR OH OR OH

The adenosinetriphosphatases (ATPases), which catalyse the hydrolysis
of ATP to ADP and phosphoric acid, fall in this category.

4. Metaphosphatases

(HPOg)™ + wHaO-^wHaPOi

{e) Deaminases
They catalyse the hydrolysis of the C — N bond of amines

\ \

— C— NH2 4- H2O -» — C— OH + NHa
/ /

(/) Deamidases
They catalyse hydrolysis of the amide bond.

-C— NH2 + H2O -^ — COOH + NH3

Examples are urease, asparaginase, and glutaminase. Glutaminase
catalyses the hydrolysis of glutamine into glutamic acid and ammonia and
asparaginase of asparagine into aspartic acid and ammonia.

ENZYMES 157

NHa

HOOC— CH— CH2

\

0==C— NHo + H2O

Asparaglne

NHo 1 ^

j Asparaginase

HOOC— CH— CH2

\

0=C— OH + NH3

Aspartic acid

NHa

HOOC— CH— CH2— CH2 -4- H2O

\

0=C— NH2

Glutamine

NH,

Glutaminase

HOOC— CH— CH2— CH2 + NH3

\

0=C— OH

Glutamic acid

158 UNITY AND DIVERSITY IN BIOCHEMISTRY

(g) Deamidinases

These enzymes catalyse the hydrolysis of amidine bonds. The most
well-known of them is arginase catalysing the hydrolysis of arginine to
ornithine and urea. Its molecule contains manganese.

HoN

NHo

\
HN— C NH CH.

CH2 CHo

-CH COOH

Arginine

+ H,0

H,N

Arginase

NH2

\
HN— C OH+H2N

CH., CHo

CHo CH COOH

Or

nithine

HoN

\
C=

=0

/

H,N

Urea

B. Phosphorylases

They catalyse the transfer of a transferable group, not to water, but to
phosphoric acid. They are transglucosidases whose acceptor is phosphoric
acid. The general formula for the transfer reaction is as follows :

(C6Hio05)„ + H3PO4 ^ CcHnOs— OPO3H2 + (CeHaoOs)™-!

Glucose- 1 -phosphate

The phosphorylases are everywhere present in the biosphere. An enzyme
of this type (phosphorylase-a) has been obtained crystalline from rabbit
muscle. In the presence of an excess of orthophosphate the reaction occurs
in the direction of phosphorolysis, whilst in the absence of orthophosphate
or in the presence of an excess of glucose- 1 -phosphate, the synthesis of
amylose takes place. The enzyme is also called glucose- 1 -phosphate ->-
amylose-transglucosidase.

From various microbial sources have been isolated a sucrose phos-
phorylase (sucrose -> orthophosphate transglucosidase) and a maltose
phosphorylase (maltose -> orthophosphate transglucosidase).

ENZYMES 159

C. Transferring Enzymes (Transferases)

Assembled in this group are the transferring biocatalysts having acceptors
other than water or phosphoric acid. They catalyse the transfer of a group
from one compound to another, and, in certain cases, they also transfer
the energy of the bond to which the group was attached. This point is
particularly important in biochemical energetics.

D— X + E ?^ D— X— E ^ D + E— X
E— X -f A ^ A— E— X ^ A— X + E

As the equations show, when the transferred portion X combines with the
enzyme E before passing to the acceptor A, the energy of the D — X bond
is not dissipated but is also transferred with little energy loss. In such a case
the system is a reversible one and there is only a small change in AF.

(a) Transphosphorylases

( Transphosphatases, Phosphokinases)

These important enzymes are numerous and universally present in the
biosphere. The Lohmann enzyme or creatine-phosphokinase has been
known for a long time. It catalyses the transfer of a phosphoric acid
residue from adenosine triphosphate to creatine (Lohmann reaction).

NH

il
ATP + H2N— C— N— CH2— COOH ^

CH3

Creatine

O NH

II !i

ADP -^ HO— P— NH— C-N— CH— COOH

OH CH3

Hexokinase transfers a phosphate group from ATP to glucose with the
formation of glucose-6-phosphate, and fructohexokinase performs the
same transfer to fructose forming fructose- 1 -phosphate. The trans-
phosphorylases can be divided into several types according to the
magnitude of AF for the transfer reaction.

Two examples will illustrate this.

Let us take the case of hexokinase which transfers a phosphate residue
from ATP to glucose with the formation of ADP and glucose-6-phosphate.
The hydrolysis of the pyrophosphate linkage of ATP gives 12,000 calories
and that of glucose-6-phosphate only yields 2,000-4,000 cal. During the

160 UNITY AND DIVERSITY IN BIOCHEMISTRY

course of the transfer there will be a AF of the order of 9000 cal. The
position of equilibrium is such that there is almost complete conversion of
glucose into its phosphate and consequently glucose-6-phosphate is not
readily able to give up its phosphate to ADP. The situation is quite different
when the transfer takes place with a small value of — AF as is the case in
the Lohmann reaction. ATP and phosphocreatine are molecules containing
an energy-rich bond and equilibrium between them will be established
when their concentrations are of about the same order.

Among the transphosphorylase systems in which energy-rich bonds are
involved and the —AF is small (so that the reaction is readily reversible)
we may list the following :

Creatine phosphokinase ATP+creatine ^ ADP+phosphocreatine

Arginine phosphokinase ATP + arginine;=±ADP + phosphoarginine

Myokinase ATP+AMP ^ 2 ADP

Phosphoglyceric phosphokinase ATP + 3-phosphoglyceric acid :^

ADP + 1, 3-diphosphogIyceric acid

Conversely, the following systems whose —AF values are high will be
practically irreversible :

Hexokinase ATP+gIucose^ADP+G-6-P

Fructohexokinase ATP-f-f ructose— >ADP+F— i — P

Phosphohexokinase ATP+F— 6— P-^ADP+F— l,6-PP

Galactohexokinase ATP+galactose-»ADP+Gal.— l — P

Glucose- 1 -phosphokinase aTP-|-G— i— P-^ADP+G— 1,6— PP

(b) Transaminases

These enzymes were discovered in 1930 by D. M. Needham, they
catalyse transfers of the following general type :

RCH— COOH + R'CO— COOH - RCO— COOH + R'CHCOOH

I I

I I

NHo NH.

An example is the glutamate-aspartate transaminase which catalyses the
reaction

glutamate + oxaloacetate ^ a-ketoglutarate + aspartate, and also
glutamate-alanine transaminase

glutamate -|- pyruvate r^ a-ketoglutarate + alanine.

There also exist transaminases which catalyse the transfer of an amino
group from amino-purines to a-ketoglutaric acid. Pyridoxal phosphate is
the coenzyme for transaminations between amino acids and keto-acids and
also between amino-purines and keto-acids.

ENZYMES 161

(c) Transpeptidases
The general reaction catalysed by transpeptidases is the following :

RCO— HNCHCO— NHR" + NH.X ^

R'

RCOHNCHCO— NHX + NH2R"

Certain peptidases catalyse these transpeptidations e.g. papain and
tryspin.

(d) Transmethylases

In general, in the biosphere, methyl groups can be transferred from a
given donor to a given acceptor in the following way:

R— CH3 + R'— H ^ RH + R'— CH3

An example of a catalyst for transmethylation is nicotine-methyl-
transferase which catalyses the reaction :

active methionine + nicotinamide ■i=i- N-methylnicotinamide

{e) Transacylases

These are enzymes catalysing the transfer of acyl residues. Certain
enzymes catalyse the transfer of the acyl residue from its combination with
CoA (cofactor for transacylations) to an acceptor.

Example : choline acetylase, catalyses the reaction

acteyl-CoA + choline i? acetylcholine + CoA

(/) Transadetiylases and Transuridylases

These are enzymes which catalyse the transfer from a given donor to a
given acceptor of a purine or pyrimidine nucleotide residue. An example
is that ATP -^ nicotinamide — mononucleotide — transadenylase, catalysing
(in presence of Mg++) the reaction:

ATP + nicotinamide mononucleotide ^ DPN + pyrophosphate
or UTP + glucose- 1 -phosphate ^ UDP-glucose + pyrophosphate

[g) Transketolases {Glycolaldehyde- Transferases)

They catalyse the transfer of a glycolaldehyde residue, for example in
the reaction :

ribulose-5 -phosphate + ribose-5 -phosphate

^ 3-phosphoglyceraldehyde + sedoheptulose-7-phosphate

M

162 UNITY AND DIVERSITY IN BIOCHEMISTRY

(h) CoA Transferases

These catalyse the transfer of CoA from an acyl-CoA to a series of fatty
acids e.g. the microbial CoA transferase catalyses the reaction :

acetyl-CoA + propionate ^ propionyl-CoA + acetate

or the CoA transferase from animals catalyses the reaction :

succinyl-CoA + acetoacetate ^ acetoacetyl-CoA + succinate

(/) Other Transferases

Other groups of transferases, which have been little studied up to the
present, are the trans-sulphurases, transglutamases, transaspartases, etc.

D. OXIDO-REDUCTION EnZYMES
(OXIDOREDUCTASES, ElECTRONTRANSFERASES)

These are transferases of a particular type, transferring electrons
accompanied by protons (transhydrogenases) or electrons alone (trans-
electronases) from a donor to an acceptor. If the acceptor is oxygen the
transhydrogenase or transelectronase is called aerobic. If the acceptor is
another type of molecule, they are called anaerobic. Here are some examples
of these four types :

(a) Anaerobic transhydrogenases

H H

/ /

R + R' ^ R + R'

\ \

H H

e.g. DPNH -^ pyruvate transhydrogenase (lactic dehydrogenase)

DPNH -^ aldehyde transhydrogenase (alcohol dehydrogenases).

{h) Aerobic transhydrogenase

H

/
R + O2 ^ R + H2O2

\
H

e.g. j8-glucose -» Oa-transhydrogenase or glucose-oxidase

Xanthine -> Og-transhydrogenase or xanthine-oxidase.

ENZYMES 163

(c) Anaerobic transelectronases

R + R'+ ^ R+ + R'

e.g. DPNH -> cytochrome-t-transelectronase or DPN-cytochrome-
reductase.

(d) Aerobic transelectronases

R + O2 ^ R+ + Or
e.g. Cytochrome oxidase or cytochrome-^g.

(a) Anaerobic Transhydrogenases
1. Enzymes Specific for the Pyridine Nucleotides
The reaction which is catalysed is :

DPN (or TPN) + RHo^ DPNH (or TPNH) + R
or more correctly

DPN+ (or TPN+) + RHg^ DPNH (or TPNH) + R + H+

Here are a few examples of reactions catalysed by specific anaerobic
transhydrogenases

a-glycerophosphate

glycerol

glucose

lactic acid

ethanol

glucose-6-phosphate
2 SH-glutathione

+ DPN+ ^ DPNH

+
phosphodihydroxyacetone
glyceraldehyde
gluconic acid
pyruvic acid
acetaldehyde

+ TPN+ ^ TPNH

+
6-phosphogluconic acid
glutathione-S-S-glutathione

2. Enzymes of Still Undefined Specificity

The nature of the specific acceptor is unknown for choline-dehydro-
genase, thiamine-dehydrogenase, and succinic dehydrogenase which is a
metalloflavoprotein containing iron.

{b) Aerobic Transhydrogenases

The transfer of hydrogen takes place, in the presence of these enzymes,
to oxygen forming HgOa- The majority of these enzymes contain a flavin
prosthetic group (FMN or FAD), but the presence of this grouping in

164 UNITY AND DIVfeRSITY IN BIOCHEMISTRY

every enzyme of this type has not been proved. In a number of cases, the
aerobic transhydrogenases are metalloproteins containing a metal (Fe, Cu
or Mo) in addition to the flavin nucleotide.
Here is a representative Ust of these enzymes :

1. (with FMN)

TPNH -^ Og-transhydrogenase (see "yellow enzyme");
L-amino acids -^ Og-transhydrogenease (L-amino acid oxidase).

2. (with FAD)

Aldehyde -^ Og-transhydrogenase (aldehyde — oxidase) ;
Xanthine -^ Og-transhydrogenase (xanthine — oxidase).

(c) Anaerobic Transelectronases

Some enzymes of this type have a flavin prosthetic group, others a haem
prosthetic group.

In the first category are the metalloflavoproteins (Fe) such as DPN-
cytochrome reductase and TPN-cytochrome reductase, enzymes which
Hoffmann-Ostenhof suggests should be called DPNH -^ cytochrome-
c-transelectronase and TPNH -^ cytochrome-c-transelectronase,

respectively.

Among the anaerobic transelectronases containing haem derivatives are
the cytochromes, described on page 119.

{d) Aerobic Transelectronases

Cytochrome oxidase or cytochrome-flg is an example of this type of
enzyme (see p. 120). It is highly specific for the transport of electrons from
cytochrome-c to oxygen. The phenolases (see p. 123) are also aerobic
transelectronases, transporting electrons from copper to oxygen.

E. Hydroperoxidases
(See page 120).

F. Lyases and Synthetases

This name applies to enzymes catalysing reactions of the type

A +B ^C

whilst the enzymes of the preceding categories (A, B, C, D, E) catalysed
reactions of the type

A +B ^C +D

Among the lyases and synthetases, some break or form a C — C bond,
these are the carboxylases or carbosynthetases, whilst others split or form
C — N or C — S bonds. Among the lyases we have the hydrases and de-
hydrases, enzymes which add or remove water.

ENZYMES 165

Pyruvic carboxylase, whose role in alcoholic fermentation is an important
one, contains thiamine pyrophosphate (TPP) as its prosthetic group and
the presence of Mg++, or in its absence, of Mn++, is necessary for its action.
This enzyme catalyses the reaction

CH3COCOOH -> CH3CHO + CO2

It is found in vegetable tissues and in bacteria but not in animal tissues.
Nevertheless, in animals and in certain micro-organisms, there is a system
for the oxidative decarboxylation of pyruvic acid which requires a series
of cofactors (CoA, DPN, TPP and thioctic acid) ; the initial reaction is as
follows :

CH3COCOOH + DPN+ -> CH3CHO + DPNH + CO2

The amino acid decarboxylases are universally distributed. They catalyse
the general reaction

RCH(NH2)C00H -^ RCH^NHa + CO2

Each of these enzymes is specific for a definite amino acid, their coenzyme
is pyridoxal phosphate attached to the enzyme protein via its phosphate.
The ketonic acid decarboxylases fall into two groups, the a-keto-
decarboxylases and the /3-ketodecarboxylases. They catalyse respectively
the follovking reactions:

RCOCOOH -> RCHO + CO2

RCOCH2COOH -> RCOCH3 + CO2

A further type of carboxylase are the triosephosphate-lyases which,
without the intervention of water, decompose hexose phosphates into a
molecule of triose phosphate and some other molecule.

Such, for example, is aldolase or FDP-triosephosphate-lyase. Universally
present in the biosphere, it catalyses the reaction

F-1, 6-PP ^ 3-phosphoglyceraldehyde + phosphodihydroxyacetone

Enolase is an example of the dehydrase type of enzyme. It is universally
distributed and catalyses the reaction

H H H

I I I
— C— C— ^ — C=C h H2O

OH OH OH

Aconitase is another hydrase, widely distributed, catalysing the reaction

- H2O + H2O

Citric acid ^ m-aconitic acid ^ L-isocitric acid
+ H2O - H2O

166 UNITY AND DIVERSITY IN BIOCHEMISTRY

G. ISOMERASES AND RaCEMASES

Racemases catalyse the conversion of an optically active substance into
its racemate. An example is alanine racemase

D-alanine ^ L-alanine
glutamic racemase

D-glutamic acid ^ L-glutamic acid
and mutarotase

a-glucose ^ ^-glucose
Isomerases catalyse molecular rearrangements, either by modifying the
structure of part of the molecule, or by displacing a part of it. To the first
class belong the following enzymes :

Reaction catalysed
Galactowaldenase Gal-l-P ^ G-l-P (cofactor : uridine-

diphosphate-glucose)
Phosphoribose — isomerase Ribose-5-P ^ ribulose -5-P

Triosephosphate^ — isomerase Dihydroxyacetone-P ^ 3-phospho-

D -gly ceraldehy de
To the second class belong :

Phosphoribumutase Ribose-1-P ^ ribose-5-P

Phosphoglucomutase G-l-P ^ G-6-P (cofactor : G-1, 6-PP)

Phosphoglyceromutase 2-phospho-D-glycerate ^ 3-phospho-

D-glycerate
(2-phospoglyceric acid) (3-phospho-

glyceric acid)
(cofactor : 2, 3-diphosphoglyceric acid)

IV. KINETICS

The velocity of an enzymatic reaction can be measured by following the
change in concentration (increase or decrease) of one of the substances
involved in the catalysed reaction, either a reactant or a product of the reaction.

For example, consider the hydrolysis of the amide groups of urea or
carbamide CO(NH2)2, a hydrolysis catalysed by a specific enzyme (the
amidase named urease) :

CO(NH2)2 + H2O -> CO2 + 2NH3

Having fixed the conditions (pH, temperature, urea concentration, urease
concentration), we may remove samples of the solution, at definite intervals
of time, inactivate the enzyme and determine, say, the ammonia.

Now if we plot these quantities of ammonia nitrogen as ordinates against
time intervals as abscissae, we shall obtain the graph shown in Fig. 31.
From the graph we can obtain the reaction velocity in moles/1. /sec. It is
the initial reaction velocity which is of interest, since as the reaction pro-
gresses the conditions change as a result of the disappearance of the
substrate and the accumulation of the reaction products, etc.

ENZYMES

167

Ammonia

nitrogen

(mg.)

Minutes
Fig. 31 — Ammonia production during the hydrolysis of urea in the presence of urease.

A. Influence of Enzyme Concentration

If we vary the concentration of the enzyme present at the beginning of
the reaction and measure the initial reaction velocity for each different
concentration, we shall find, in general, that the velocity increases linearly
with the concentration of the enzyme : the reaction is therefore first order
with respect to the enzyme.

Units of
proteolytic
activity 2

00 cc.

(volume of solution containing 1ml of trypsin solution.)

Fig. 32 (Northrop). — Influence of the enzyme concentration on the speed of hydrolysis.
Curve A : pure trypsin, proportionality between hydrolysis and tr>'psin concentration.
Curve B : ordinary impure enzyme; the speed of hydrolysis is not proportionately increased

as the enzyme concentration is increased.

168

UNITY AND DIVERSITY IN BIOCHEMISTRY

In the case of peptidases, doubt has long been cast on the validity of this
finding : it appeared that the speed of hydrolysis increased less rapidly
than the enzyme concentration. Northrop has provided an explanation of
this phenomenon, in the case of pepsin and trypsin, by showing that the
effect is due to the presence of impurities exercising an inhibitory action
on the enzyme,

B. Influence of pH

If the pH is varied, one normally observes that the initial velocity passes
through a maximum which is called the "optimum pH". This is a con-
sequence of the protein nature of the enzyme. The phenomenon is ex-
plained by postulating that the active part of the enzyme consists of a
— C00~ group and a — HN3+ group associated through their charges.
Changes in pH consequently change the concentration of the active form
of the enzyme in the solution.

H+
OH-

NH2— COO-^ NH3+— COO- ^ NH3+— COOH.

Inactive enzyme

H+
OH-

Active enzyme

Inactive enzyme

Activity

10 n pH

Fig. 33 (Michaelis and Davidson) — Activities of j8-fructosidase (invertase)
and trypsin at difTerent pH values.

ENZYMES 169

C. Influence of Substrate Concentration

When the substrate concentration is varied, in general it is found that the
initial velocity changes with the concentration of substrate in a linear
manner. In other words the reaction is first order with respect to the
substrate.

However this is no longer the case when the substrate concentration is
greater than a certain value : the initial velocity may then become indepen-
dent of the substrate concentration. In order to explain this, it is postulated
that the reaction takes place in two stages, a combination of the enzyme
with the substrate (E + S ^ X), and a decomposition of the enzyme-
substrate complex with regeneration of the enzyme (X ^ P + E).

^^-^^ O O^O o OJD O y

o%ooOo o

qO o

o

Fig. 34 — Enzyme-substrate relations at a low, and at a high, concentration of substrate.

When the amount of substrate is small, a little of the enzyme combines
with the substrate but many free enzyme molecules remain. The quantity
of the enzyme-substrate complex formed in unit time is proportional to the
concentration of the substrate, and the initial velocity, too, is proportional
to this concentration. But if, relative to the enzyme, there is plenty of
substrate, the enzyme will be present wholly in the form of the complex,
such that the addition of further molecules of substrate does not alter the
situation as far as the concentration of the enzyme-substrate complex is
concerned (Fig. 34).

D. The Michaelis Constant ^

Let us represent by [E]q the total concentration of the enzyme and by [E]
the concentration of the free enzyme. [S] will represent the concentration

170 UNITY AND DIVERSITY IN BIOCHEMISTRY

of the substrate and [X] the concentration of the enzyme-substrate com-
plex. When the enzyme-substrate reaction is at equilibrium :

\X}

= K (equilibrium constant for the enzyme-substrate reaction) (1)

[E] [S]

Each molecule of the enzyme-substrate complex contains a molecule
of enzyme and a molecule of substrate so

[E], = [E] + [X] (2)

Equation (1) can therefore be written :

[X]

{[E]o - [X])[S]

= K (3)

Now, it is the concentration of the complex [X] which governs the
velocity of the reaction

V = k,[X] (5)

The dissociation of the complex X is veiy far from the position of equili-
brium and we may neglect the velocity of the reaction

S +E-^X
From (4) and (5),

KKjEUS]

'' = TTk[S] ^^^

K (the equilibrium constant of the enzyme-substrate reaction) is often
replaced by its reciprocal 1 jK = Km, or the Michaelis constant.
If we replace i^ by 1 /Km, equation (6) becomes

^ k,IK„>[EUS]

l+{llKm)[S] ^^

It can be shown that Km, the Michaelis constant, which has the dimen-
sions of concentration (moles/1.), is in fact the substrate concentration
corresponding to a value of half the maximum velocity (Fig. 35). Know-
ledge of the Michaelis constant for a given enzymatic reaction, allows v to
be calculated for any value of [S], provided that Ag is known.

ENZYMES

171

If we compare K = 1 1 Km and K = [X]I[E] [S], we see that Km is the
value of [S] for which [X] = [E], that is to say it is the value of [S] at
which half the enzyme is combined with the substrate, and at which con-
sequently {v = /?2[^]), the reaction velocity is half the maximum velocity
for that enzyme concentration (Fig. 35). Km is measured, like S, in moles/1.

msx

Molar

Fig. 35 — Current method of measuring the Michaehs constant, v = reaction velocity in
moles per min. Vmax = maximum velocity (the reaction velocity increases with the con-
centration of substrate but not proportionately). When all the enzyme molecules have
formed the enzyme-substrate complex, a further addition of substrate no longer increases
the velocity. The maximum velocity has been reached. [<S] = molar concentration of the
substrate (the abscissa scale is logarithmic). Km = 0-02 in this example.

V. THE MECHANISM OF ACTION OF ENZYMES
AND COENZYMES

A. Enzymes

At the present time, our views on the manner in which enzymes evade
the necessity for molecular activation are very hypothetical.

Barnard and Laidlaw for exam.ple have attempted to explain the mode
of action of hydrolases by a "bifunctional" effect. One can discuss catalysis
with respect to amino and carboxyl groups without implying that these
groups are necessarily the ones which are responsible for the catalytic
action.

172

UNITY AND DIVERSITY IN BIOCHEMISTRY

The enzyme molecule bears — NH3+ and — COO~ groups. The former
may lose a proton and the latter can accept a proton. It can be postulated
that when an ester is hydrolysed by an esterase, the — NH3+ grouping
approaches the alcohol oxygen and donates a proton to it. Simultaneously,
the — C00~ approaches the carbon of the carbonyl group. Also, it must be
postulated that a water molecule remains sandwiched between the — C00~
and the C atom, and that the — C00~ tends to draw to itself a proton from
the water molecule at the same time assisting the 0H~ ion to approach
the carbon of the carbonyl group. The picture is as follows :

O

II
R— C— O— R'

H— O

I
H H

o-

0=C N+

o-

R— C 0+— R'

I
H- H

I
O

I
0=C N+

Enzyme

Enzyme

The enzyme-substrate complex thus formed can then split in the follow-
ing manner :

R— C

OH 4- H— O— R'

H

O

I
0=C N

Enzyme

This proposed mechanism is based upon the idea that a protein is a semi-
conductor, in which, as in a metal, certain electrons are not strictly local-
ized to definite points in the macromolecule, but are, to a certain degree,

ENZYMES

173

free to move around in the space occupied by the macromolecule. Accord-
ing to this conception, the electrons are poured into a "pool" covering the
macromolecule and the tributaries of this pool are the hydrogen bonds
between the polypeptide chains. In this context, an enzyme is a semi-
conductor of greater efficiency than an ordinary non-enzymatic protein.

In the explanation of the hydrolytic action outlined above, the enzyme
is considered as a conductor of electrons through which an electron flow
takes place, so modifying the distribution of electrons that certain bonds
are ruptured.

For the hydrolysis of a peptide, the picture will be as follows :

O H

o-

H

R— C— N— R'

H— O

I
H H

o-

I
0=C N+

Enzyme

R— C-

I
HO+

I
H-

I
O

o=c

-N— R'

H

N-

Enzyme

i

O

R— C 4- R'NHz

OH

H

I
O

I
0=C N

Enzyme

B. Coenzymes

When the enzyme requires a coenzyme, we know a little more about the
mechanism, at least as far as the latter is concerned.

If, for example, we consider the lactic dehydrogenase system, we may

174

UNITY AND DIVERSITY IN BIOCHEMISTRY

postulate that the reaction begins by the fixation of lactic acid and the
coenzyme DPN+ on the enzyme protein molecule (apoenzyme).

Lactic acid

DPN

Apoenzyme

A proton is then lost by the lactic acid to the solution and, simul-
taneously, an electron passes from the lactic acid to the apoenzyme.

1

0

1

CHj CHOH COOH

DPN CH3 CH COOH

DPN

iX

Apoenzyme

Apoenzyme

The electron travels along the apoenzyme until it reaches the coenzyme
where it combines with a proton furnished by the solution and the DPN+
is reduced to DPNH.

I

O

CH, CH COOH

H+

I

DPN

O

I

CH, CH COOH

H-DPN

Apoenzyme

Apoenzyme

The role of pyridoxal phosphate as a coenzyme in the decarboxylation
of amino acids can, according to Mendeles, Koppelman and Hanke (1954),
be explained by the following scheme :

. coo-
* I

H-C=0 HsN-C-H

HO

HsC

sC t^Vj.

CHjOPOaHz

+H2O

COO"
I
H— C=N— C-H
I

-H,0 J^ R

H

-CO,

+COa

H-C=N-C— H
I
R

H

H

H— C=0+H8N -C— H

— HoO

+H2O

H

H-C=N.-C— H

N
H

-H*

+H*

H-C-N=C— H

A k

N'
H

ENZYMES

175

Another example in which the intervention of a coenzyme is explained,
is the case of the conversion of G — 1 — P into G — 6 — P. Here, G — 1, 6 — PP
plays the part of coenzyme. Unlike the case of DPN cited previously, this
coenzyme does not function as a second substrate, but on the contrary
it is constantly reformed from the substrate. In effect a cycle is operating
in which G — 6 — P accumulates.

G— 1, 6— PP + G— 1— P ^ G— 1, 6— PP + G— 6— P

An exactly analogous mechanism is that in which 2, 3-diphosphoglyceric
acid acts as the coenzyme for the phosphoglyceromutase system (trans-
formation of 2-phosphoglyceric acid into 3-phosphoglyceric acid or the
inverse) (Sutherland, Posternak and Cori, 1949) :

COOH

COOH

O

o

COOH

I
HCOH

HC— O— P— OH HC— O-P— OH

I +
OH

O

H2C— O— P— OH H2COH

OH

+

OH

2, 3-diphospho-
glyceric acid

2-phospho-
glyceric acid

I II

HoC-O-P— OH

OH

3-phospho-
glyceric acid

COOH

I

I o

I II

HC-O-P-OH

I
OH

O

II
H2C-O-P-OH

I
OH

2, 3-diphospho-
glyceric acid

Another coenzyme whose action has been clearly elucidated is coenzyme
A, the coenzyme for acetyl transfer. Its sulphydryl group is capable of being
alternately acetylated and deacetylated in the same way as DPN can be
alternately oxidized and reduced.

O

o

R— C— OH+HS— CoA ^ R— C

Acyl

S— C0A+H2O

To illustrate the mechanism of CoA intervention, let us take for an
example the acetylation of an amine, the energy being supplied by ATP.
Thermodynamically, we may have

ATP + CH3— COOH ;=^ CH,CO--P +ADP
CH^CO'-P H- NH.— R -^ CH3CO— NHR + P

176 UNITY AND DIVERSITY IN BIOCHEMISTRY

Nevertheless, if we consider the two sets of reactions, A and B, below, we
see that either of them is faster than that written above. Both introduce
CoA, which acts as the catalyst.

A) CHaCO'-P + PnA-^SH^CoA— S^COCH. -4-P ^

CoA — S-COCHa + NH2— R -^ CH3CO— NHR -j- (CoA-SH)

B) ATP + CoA -^SH ^ CoA-S-PP + AMP ^X^

CoA— S-PP + CH3COOH ^ CoA — S-COCH3 + PP \
C0A-S-COCH3 + NH2-R -^ CH3CO-NHR + (CoA— SH)

We see that at the end of the reaction, the catalyst remains intact and
ready to function again.

Besides DPN, a number of other coenzymes of the oxido-reduction type
act by reason of their ability to be alternatively oxidized and reduced.
These are the coenzymes of hydrogen transport : TPN, FMN, FAD and
thioctic acid, whose structure we have already considered.

The few examples above give some idea of the variety of mechanisms
of coenzyme action, an action which operates in conjunction with the
activation by the enzyme macromolecule itself. The mechanism of this
activation is one of the most important problems facing modern bio-
chemistry.

REFERENCES

Laidler, K. J. (1954). Introduction to the Chemistry of Enzymes, McGraw Hill

New York.
HoFFMANN-OsTENHOF, O. (1954). Enzymologie, Springer, Wien.
Mehler, a. H. (1957). Introduction to Enzymology, Academic Press, New York.
DixoN, M. and Webb, E. C. (1958). Enzymes, Longmans, Green and Co, London.

PART THREE '
CHEMICAL REACTIONS IN THE BIOSPHERE

N

J

INTRODUCTION

The biosphere is the seat of a continuous transformation of matter and
energy. Despite influences tending constantly to convert the chemical
elements into the highly oxidized state characteristic of inanimate matter,
this activity maintains the living material and replaces it continuously
throughout its more or less prolonged, but finite, life-span. The metabolism
of the organisms Vv^hich make up the biosphere shows certain general
characteristics common to all organisms, besides those applying only to
particular t\'pes or classes. The organism makes pyrophosphate bonds ready
for the performance of various types of cellular work by means of a chemical
machine whose essential character is everywhere the same. This machine
can be fed by the chief types of chemical structure which are present to a
high proportion in all types of protoplasm : sugars, fatty acids and amino
acids. This applies also to those organisms which receive their energy from
outside the biosphere and are called autotrophes (see Part Five). In the
autotrophes, which can build up organic macromolecules from simple
inorganic materials, and in the heterotrophes (organisms depending on a
supply of protoplasm from another organism, which can be broken down),
the supply of cellular nutrients (sugars, fatty acids and amino acids) in
most cases requires a corresponding breakdown of macromolecules formed
by the association of these nutrients. Here we have an important generaliza-
tion which has already been mentioned by Herbert Spencer in his Principles
of Biology. The macromolecules which form the plastic structure pre-
dominant in living beings should in fact have a certain stability, which will
allow them to act as a reserve for nutritional purposes and which will assure
the persistence of form, despite what we now know to be a dynamic state
which, in the course of time, renews these molecules more or less quickly,
completely or partially. On the other hand these polymers should be
endowed with a certain instability permitting them to deliver up the whole
of their monomer content to the turnover of cellular nutrients when
protoplasm is acting as food. This, we know now, is not due to the in-
stability of the macromolecules but to the presence of specific enzymes, the
hydrolases, which can cleave peptide, oside and ester bonds. There are not
very many diff"erent kinds of hydrolases, but they are found in extremely
diverse locations and employed in a variety of different ways; in fact their
study is of interest both to the biochemist and to the comparative physio-
logist. The final result of their action is the liberation of fatty acids, sugars
and amino acids, which become available to the metabolism of the cell and
form its actual nutrient.

179

180 UNITY AND DIVERSITY IN BIOCHEMISTRY

The cell is the place where an important series of reactions occur which
we shall term priming reactions since their purpose is the formation of the
energy-rich linkages of ATP. These reactions are the most important
mechanism for the provision of these energy-rich bonds. They are not
what were formerly known as catabolic reactions, but they consist of
a series of closely interrelated cycles which are more or less reversible. As
the cellular nutrients are metabolized and consumed in these cycles, a
certain number of energy-rich bonds are formed, some new molecules are
synthesized and above all, the cell is provided with a series of fragments
containing one, two or more carbon atoms. These fragments can serve as
building blocks for the construction of many different molecules which
can be incorporated into the structure and functioning of that particular
organism.

CHAPTER I

DESTRUCTIVE AND NON-DESTRUCTIVE
METHODS IN MODERN BIOCHEMISTRY

I. FROM THE WHOLE ORGANISM TO THE PURE ENZYME

Many types of biochemical research are carried out on the whole organism,
with a minimum of disturbance. This is the case, for example, when a given
compound is added to the food and the excreta is investigated for this
substance or its transformation products.

An example of important information obtained by this non-destructive
procedure is the demonstration of nitrogen equilibrium in the adult
animal, as in the classic experiments of Bischoff and Voit on the dog. If
meat is given to a dog continuously over a period of several days, and the
nitrogen excretion is measured over this time, it is found that no nitrogen
is retained. The nitrogen excreted corresponds to the nitrogen ingested;
the animal is said to be in nitrogen equilibrium.

The organism can also be subjected to experimental intervention such as,
for example, the removal of an organ. The normal mammal converts the
nitrogen of amino acids into urea, but this is no longer the case when the
organism is deprived of the liver. The amino acids and ammonia accumu-
late in the blood. Alternatively, the urea formed when the liver is present is
eliminated by the kidneys and it is the urea which would accumulate in the
blood if the kidneys were removed.

A more destructive process is experimentation using isolated or perfused
organs. Thus it is possible to introduce a substance to an organ by way of
the perfusion fluid and to observe the changes it undergoes in the organ by
studying the efferent fluid. For example, introduction of ammonia into
the liquid perfusing the isolated liver of a dog results in the appearance of
urea in the efferent liquid. Alternatively, the same experiment using isolated
goose liver would show the appearance of uric acid. It is evident that an
isolated perfused organ, whether perfused with "physiological saline" or
with blood, is in fact a highly abnormal experimental material. It is ab-
normal when we consider the supply of oxygen to the cells, the supply of
blood, the inervation of the material, etc. Even more destructive is experi-
mentation using thin tissue slices. Such slices of tissue, provided that they
have been prepared under suitable conditions, will survive for several
hours, the cells appearing to lead a normal life. The pressure of oxygen
must be sufiicient to meet the oxygen requirements of the tissue. If the

181

182 UNITY AND DIVERSITY IN BIOCHEMISTRY

medium is buffered and the slice is sufficiently thin (0-3 mm approx.) this
method is very useful. Thanks to the manometric method developed by
Warburg, the use of tissue slices has given much important information.

The next stage in the order of gradually increasing destruction is the use
of minces, breis and homogenates. Here, the cells are destroyed but the
intra-cellular particles are intact. They may even be isolated by centri-
fugation, and mitochondria are often separated in this way. However,
although all the above methods are useful and instructive, only the results
obtained with purified enzymes have been capable of giving precise infor-
mation about metabolic mechanisms. The rapid progress of biochemistry
during the course of the last few j^ears is primarily the result of the perfecting
of methods for the isolation, purification and characterization of enzymes.

If the results so obtained are clear and precise, they are evidently also
reached through the most extreme of destructive processes and the re-
constitution of the complex phenomena of metabolism from such results
might appear, and in fact does appear to certain intellects, a most far-
fetched undertaking.

As enzymology was developing, biochemistry also was shaking off the
shackles imposed upon it by chemists on the one hand, and physiologists on
the other. As the particular nature of its own problems became apparent,
biochemistry began to explore all the paths of Nature in search of their
solution, breaking open the compartments into which an out-of-date
classification of the sciences had tended to confine it. Having recourse to
recent discoveries in physics and genetics, the biochemists took the cer-
tainties obtained from the use of purified enzymes, and with indications
provided by the use of isotopes and mutant forms of micro-organisms they
developed new non-destructive methods to go with the extremely de-
structive methods of enzymology. It is due to this combination of ingenuity
and patience that numerous problems of biosynthesis, up till then obscure,
have been solved.

II. BIOCHEMICAL INVESTIGATION AND THE
USE OF ISOTOPES

By means of isotopes it is possible to mark a compound at one or more
positions in the molecule and to follow the fate of these labelled atoms. With
the introduction of isotopes of the most common elements in the biosphere
(hydrogen, carbon, nitrogen, oxygen, phosphorous, sulphur), the method
has proved most fruitful.

REFERENCES

Neilands, J. B. and Stumpf, P. K. (1955). Outlines of Enzyme Chemistry. Wiley,
New York.

DESTRUCTIVE AND NON-DESTRUCTIVE METHODS 183

According to the present view, the nucleus of an atom is formed from
protons of mass 1 and charge + 1 and neutrons of mass 1 and charge 0.
The atomic number corresponds to the number of protons and all the
atoms of a given element have the same number of protons in the nucleus.
But the total number of protons and neutrons can vary. To designate the
different isotopes of an element, its symbol is written preceded by a number
corresponding to the atomic number (i.e. to the number of protons) and
it is followed by an index corresponding to the number of nucleons
(protons 4- neutrons).

The three isotopes of hydrogen, protium, deuterium and tritium are
written as follows:

iHi iH2 iH3

and the four most important isotopes of carbon thus :

pll pl2 pi3 r>14

6^ 6^ 6^^ e^-'

Since the atomic number is the same for the several isotopes of an ele-
ment, it is often omitted, and only the number of nucleons is noted.
Certain isotopes are stable and occur naturally and they may be concen-
trated from these natural sources. The most important as far as the
biochemist is concerned, are lH^ qC^^, vN^^ and gO^*.

It is also possible to prepare artificial isotopes by bombardment with
protons, neutrons, a-particles, etc. These isotopes are radioactive and after
varying lengths of time they undergo transmutation accompanied by the
emission of electrons. The determination of stable isotopes is carried out
with the mass spectrometer and radioactive isotopes are determined by
measuring their degree of radioactivity.

If we consider a homogeneous population of radioactive atoms, a con-
stant proportion of this population will decay in any given period of time.

An interesting characteristic of each isotope is its half life, a figure
corresponding to the time required for exactly half of the total number of
atoms to decay. It is equal to infinity for the stable isotopes. It is twelve
years for tritium, 5900 years for eC^^, but only fifteen hours for iiNa24 aj^(i
20*5 minutes for qC^^.

One of the most useful applications of isotopes is in the study of meta-
bolic problems and the determination of a precursor -product sequence.
Suppose that we wish to know whether A is converted to B inside the
organism. We synthesize A; introducing atoms of an isotope, we administer
it to the organism and then we isolate the compound B after some little
time. We degrade this compound and determine the distribution of the
isotopic atoms in the molecule. This procedure often casts useful light on
the mechanism of the conversion.

Isotopes also enable us to measure the speed of synthesis or breakdown

184 UNITY AND DIVERSITY IN BIOCHEMISTRY

of a compound. In this way one can measure the turnover of a substance,
that is the rate at which its molecules are replaced (although the concen-
tration remains the same) when it is in a steady state resulting from an
equilibrium between the rate of synthesis on the one hand, and the rate of
breakdown or incorporation, on the other.

III. THE USE OF MUTANT STRAINS OF
MICRO-ORGANISMS

The name auxotrophes is given to those mutant forms of a micro-
organism which are dependent on the provision of a growth factor not
required by the natural form. At the present time, a very large number of
mutants are known which are characterized by the loss of a given enzyme,
their metabolism is blocked at the stage of a definite chemical reaction.
To define the particular reaction which is blocked, two sets of information
are required : a knowledge of the substances which the mutant can use as
growth factors, and knowledge of the substances which accumulate in the
cell. Let us suppose that A and B are two different precursors of X. If
we have a mutant which is an auxotrophe for X, which accumulates A,
and which responds by growing when B is supplied, we can deduce that
the block is situated after A and before B in the series of metabolic
reactions.

Distinction between a Possible Precursor and an
Obligatory Intermediate

To show that a given substance can serve as a precursor of a second
substance is one thing; to show that it is in fact the normal intermediate
in the organism is quite another. The study of mutants of micro-organisms
has revealed the existence of auxotrophes for each of the naturall)'^ occurring
amino acids. This illustrates very well the idea of an obligatory metabolic
pathway, at least in these organisms.

Let us once more consider the case of A and B, precursors of X in a
micro-organism. The wild strain of this micro-organism is able, as can be
demonstrated by the use of isotopes or by means of the purified enzymes,
to convert A and B into X. A single enzyme, extracted from the micro-
organism and purified, converts A into B. A mutant auxotrophic for X does

REFERENCES

Calvin, M., HEmELBERCER, Ch., REm, J. C, Tolbert, B. M., Yanknich, P. F.

(1949). Isotopic Carbon. Techniques in its measurement and chemical manipulation.

Wiley, New York.
Kamen, M. D. (1948). Radioactive Tracers in Biology. Academic Press, New York.

DESTRUCTIVE AND NON-DESTRUCTIVE METHODS 185

not contain this enzyme. In this way, by association of destructive and non-
destructive methods, both of which are indispensable, it is possible to
demonstrate with certainty that the metabolic sequence is A-B-X.

Nevertheless, it can be objected that a product accumulated by one
mutant and utilized by another mutant could very possibly be the product
of a side-reaction.

For example, consider the reaction scheme

t
A'

It is possible to imagine, if the block is between A and B, that an extract
of the wild strain of the micro-organism would covert A' into B whilst an
extract of the blocked mutant would not. Is this a reason for placing A' in
the direct metabolic sequence to X, that is for writing A ^^ A' -^ B ^' X}
No, because the isolation of enzymes enables us to demonstrate that a single
enzyme is capable of converting A into B and that, for the conversion of A'
into B, two enzymes are necessary; in addition to the first, v/e require an
enzyme catalysing the reaction: A' -^ A. This is an illustration of the
importance of studies on purified enzymes, alongside experiments using
non-destructive methods.

REFERENCES

Harris, H. (1953). An Introduction to Human Biochemical Genetics. Univ. Press,

Cambridge.
Wagner, R. P. and Mitchell, H. K. (1955). Genetics and Metabolism. Wiley,

New York.

CHAPTER II

PRIMING REACTIONS

I. GLYCOLYSIS AND THE HEXOSEMONOPHOSPHATE

SHUNT

The name glycolysis is given to the sequence of enzymatic reactions
(Embden-Meyerhof scheme) which bring about the fragmentation of
carbohydrates by a pathway which does not involve the intervention of
oxygen molecules. The enzymatic system for glycolysis is a universal one,
at least in its main outlines, although there are numerous variations differ-
ing in certain details.

The most completely understood system is that of the alcoholic fermen-
tation of glucose in the presence of yeast.

A. Alcoholic Fermentation

The fermentation of grape juice (pH around 5'0) under the influence of
yeasts growing on the surface of the grape (especially Saccharomyces
cerevisiae) and transported from one to another by insects (in particular
wasps), has been known since ancient times. The manufacture of beer and
of bread are further well-known examples of alcoholic fermentation, that
is the anaerobic breakdown of glucose with production of ethanol and COg.
It was Theodor Schwann who first showed, in 1837, that the alcoholic
fermentation of grape juice, at that time a phenomenon of some mystery,
depended on the introduction of living cells into the sweet solution. He
described yeast simultaneously with Cagniard-Latour. This great dis-
covery at once encountered the open opposition of the chemists. For them,
alcoholic fermentation was a simple chemical process, expressed by the
equation

CeHiaOe ^ 2C2H5OH + ZCO^

Liebig even went so far as to draw up with Wohler, and to publish
anonymously in Annalen der Pharmacie, a facetious article ridiculing
Schwann's views and depicting yeast as a sort of infusoria eating sugar,
excreting alcohol from the digestive tract and COg from a bladder in the
shape of a bottle of champagne. Twenty years later, in 1860, Pasteur
confirmed the views of Schwann, and the "vitalist" theory of fermentation
triumphed.

In 1897 Buchner, whilst preparing extracts of yeast for a therapeutic

186

PRIMING REACTIONS

187

purpose, wished to assure their preservation by the addition of sugar and
he made an important observation : the mixture frothed and became rich
in alcohol. Alcoholic fermentation without cells had been discovered. Our
present knowledge of this phenomenon derives from elaboration of the
results of an experiment done by Harden and Young in 1906. Buchner
had observed that if one added phosphate to yeast juice, the COg production
was increased — for him this was the result of the change in acidity.

130
120
110

100

r — 1

.Cr^

y^

ro

_..

iA

r)

)--Vtr

V'-'

/

c

A

^

80
70

CI

^

J

Y

\ }

c

!^

yOr^

H-W

f

0 ou

^,.--

,■■■'

A

r

r

50

V'

40
30
20

10

)

*i— --

-*.-

'^

-^<.

;

/

-r-^

s-*^

,

A

^

g^ '

"A

1

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 ISO

TIME IN MINUTES

Fig. 36 (Harden and Young) — Production of CO2 with time in yeast juice to which has
been added : a. glucose; b. the same mixture (25 ml of yeast juice + 25 mlof water in which
5g of glucose is dissolved) plus 5 ml of a 0-3M mixture of primary and secondary phos-
phates; c. a further addition of the same quantity of phosphates after 70 min.

Harden and Young observed that after a temporary increase in COg
evolution, following the addition of phosphate, the effect died away, only
to be renewed on a further addition of phosphate. Moreover, successive
additions of phosphate led to a production of one mole of COg for each mole
of phosphate added and the increase in the production of alcohol corre-
sponded mole for mole to the amount of CO2 produced (Fig. 36).

When Harden and Young sought the phosphate which they had added,
they found that it was not in its free form but was bound as phosphoric

188 UNITY AND DIVERSITY IN BIOCHEMISTRY

esters. They isolated fructofuranose — 1,6 — PP (Harden and Young ester)
and later other esters have been isolated from the fermentation liquor :
fructofuranose — 6 — P (Neuberg ester); glucose — 6 — P (Robinson ester)
and glucose — 1 — P (Cori ester).

In 1905, a new fundamental discovery was announced simultaneously
by Buchner and by Harden and Young. When a fermenting juice is dia-
lysed, fermentation ceases in the dialysate (termed cozymase) and in the
residue (termed apoenzyme). In the "cozymase" or "coenzyme" (the name
given initially to the dialysate), we find substances of the nature of our
"coenzymes", ATP, DPN, DPT.

The reducing action of yeast thus became the foremost topic of interest.

Neuberg carried out another important experiment by adding bisulphite
to the fermenting mixture. A precipitation of the bisulphite complex of
acetaldehyde occurs and glycerol accumulates (one mole per mole of the
bisulphite compound). Neuberg concluded that fermentation takes place
by a decarboxylation of pyruvic acid forming acetaldehyde, and that the
glycerol contains the hydrogen which, in fermentation, reduces the acetal-
dehyde to alcohol. But what was the substance which on hydrogenation
yielded glycerol? This could only be a triose. For a long time it was believed
to be methylglyoxal, but we now know that it is an equilibrium mixture of
phosphoglyceraldehyde and phosphodihydroxyacetone. We know today
how these trioses are produced from glucose. Alcoholic fermentation takes
place in the following stages :

1. In the presence of hexokinase, a molecule of ATP reacts with glucose
to form G — 6 — P. This reaction is a transphosphorylation having a large
negative AF. Equilibrium is as follows :

ATP + glucose -> ADP + G— 6— P

1% 99%

Hence it is an almost irreversible reaction in which the energy of the ATP
bond is lost almost completely.

2. In the presence of phosphoglucoisomerase, G — 6 — P is transformed
rapidly into F — 6 — P and the equilibrium of this reversible reaction is :

G— 6— P ^ F— 6— P

70% 30%

3. Phosphofructokinase, in the presence of Mg++, and very specifically,
catalyses the transfer of the terminal group of ATP to F — 6 — P with
formation F — 1, 6 — PP. Here also, as in 1, we have a transphosphorylase

PRIMING REACTIONS

189

system of high negative JF, such that the energy of the energy-rich bond
of ATP is not conserved and the reaction is irreversible.

F— 6— P + ATP -> F— 1,6— PP + ADP

4. The formation of phosphotriose, the substrate for glycolysis, takes
place by the splitting, in the presence of aldolase, of F — 1,6 — PP into a
mixture of two triosephosphates. Equilibrium between the hexose-
phosphate and the mixture of triosephosphates is established in the cell.

CH20PO(OH)2
Phosphodihydroxyacecone |

CO

F - 1,6 - PP;=i + CH2OH

aldolase \[

J-phosphoglyceraldchyde CHO

I
CHOH

96 7o

CH20PO(OH)2

} 4 Vo

89 %

11 %

70

5. In the course of reactions 1 to 4, a molecule of glucose has been
transformed into a mixture of F — 1,6 — PP and two triosephosphates.
Now occurs the first anaerobic oxido-reduction (see p. 142) in which, in
the presence of triosephosphate dehydrogenase and its coenzyme DPN, an
internal oxido-reduction takes place forming 1,3-diphosphoglyceric acid,
a molecule containing an energy-rich acylphosphate bond.

H 0-P0(0H)2

/ /

c=o c=o

I + DPN+ + H3PO, - I -\- DPNH +H+

CHOH CHOH

CHoOPOCOH), CH20PO(OH)o

3-phosphoglyceraldehyde 1,3-diphosphoglyceric acid

6. This reaction causes the F — 1,6 — PP to dissociate into triose phos-
phates, since it uses up one of the products of that reaction. Reaction 5
itself is pushed completely to the right (although it is a reversible reaction)
by the fact that, in the presence of the very specific enzyme 3-phospho-
glycerate phosphokinase, the energy-rich compound 1,3-diphosphoglyceric
acid transfers its energy-rich bond to ADP to form ATP. The phos-
phokinase acts almost at equilibrium so that the energy-rich bond is
transferred with little loss (1 acylphosphate bond -^ 1 pyrophosphate
bond) (low —AF).

190 UNITY AND DIVERSITY IN BIOCHEMISTRY

7. After the first transfer of phosphate described in 6, 3-phospho-
glyceric acid is left. In the presence of phosphoglyceromutase and
2,3-diphosphoglyceric acid as coenzyme (see p. 175) an isomerization to
2-phosphoglyceric acid occurs.

8. It is at the level of 2-phosphoglyceric acid that the second internal
oxido-reduction appears. In the presence of enolase and Mg++, a molecule
of water is removed and a molecular rearrangement generates (see p. 143)
an energy-rich bond.

CH.OH CH2

I II

HC— OPO(OH)2 ^ C— 0'-P0(0H)2 + H2O

I I

COOH COOH

2-phosphoglyceric acid phosphoenolpyruvic acid

9. The phosphoenolpyruvic acid is the subject of the second transfer
of phosphate, in the presence of pyruvic phosphokinase, a phosphokinase
acting almost at equilibrium (low AF) the transfer is brought about with
conservation of the energy-rich bond.

COOH COOH

I I

COPO(OH)o -f ADP?=^ COH + ATP

CHo

CH2

phosphoenolpyruvic acid

enol-pyruvic acid

11

COOH

CO

I

CH,

pyruvic acid

10. The pyruvic acid, in the presence of pyruvic carboxylase (the
prosthetic group of which is DPT) and Mg++, is decarboxylated to
acetaldehyde.

Mg++
CH3— CO— COOH -> CH3— CHO + CO2

DPT

11. When the 1,3-diphosphoglyceric acid is formed, in reaction 5,
DPN+ is reduced. The DPNH which results is first of all dehydrogenated
by phosphodihydroxyacetone producing 3-phosphoglycerol, which is then
hydrolysed by a phosphatase to form glycerol. This is the explanation of

PRIMING REACTIONS

191

the production, first noted by Pasteur, of small amounts of glycerol during
alcoholic fermentation. But acetaldehyde is more strongly oxidizing than
phosphodihydroxyacetone. As long as it is present it will take hydrogen
from the DPNH to form ethanol.

Glycogen (or starch)

phospliorylju-

Galactose

+ ATP
gjl.ictokimse

Fructose

_ _, gjliciowjlJinuc

G— 1— P — Galactose— 1—?

UDPG

G-1,6 — FP

Hcxose menophosphate shur.t

dipliosphofriictosophosphat.ise

F— 1,6— PP

I*hosphodih\'clrox)'acetonc

Pl-.ospho^lyceraldchydc

Fig. 37 — Preludes to glycolysis.

B. Preludes to Glycolysis

Glycolysis is essentially the passage of F — 1,6 — PP to pyruvic acid by
the intermediary of a splitting into two triosephosphates, two oxido-
reductions and two transfers of phosphate groups. It is an anaerobic
oxido-reduction of trioses, at substrate level, with formation of two energy-
rich bonds per molecule of triose.

Glycolysis, defined as above, is very common in the biosphere. There
are numerous variants of the beginning of the process. The entry into
the pathway varies from one carbohydrate to another, as Fig. 37
sufficiently illustrates.

C. The Hexosemonophosphate Shunt
(Pentose Cycle)
Although it is true that the Embden-Meyerhof scheme traces the most
general form of the start of carbohydrate catabolism, there exists an alter-
native route, oxidative in nature, which with a fragment of the glycolysis

192 UNITY AND DIVERSITY IN BIOCHEMISTRY

chain constitutes a cycle resulting from the attachment of a shunt, one end
on G — 6 — P and the other on F — 6 — P and the triosephosphates. The
multi-enzyme system of the hexosemonophosphate shunt (HMS) is some-
times called the pentose cycle because it contains mechanisms for the
formation of pentoses either by decarboxylation of hexoses or from
phosphoglyceraldehyde. The cycle is quite widely found in the biosphere,
but its relative importance compared to glycolysis is extremely variable.
The different tissues of an organism differ in this respect. In mammals, for
example, glycolysis is predominant in the muscles and the hexosemono-
phosphate shunt in the liver.

Knowledge of the different reactions which have been carried out in
vitro with purified enzymes, reactions which are collected together in the
scheme shown in Fig. 38, give experimental confirmation to the ideas
summarized in this scheme. It does not exclude the existence of other, as
yet unknown, pathways.

The cycle contains two oxidations, each coupled with TPN (and not
DPN which in general is the coenzyme required in glycolysis). Glycolysis
is inhibited by fluoride and iodoacetate or bromacetate, the first affecting
enolase and the second triosephosphate-dehydrogenase.

In 1936, Lipmann found that an extract of yeast continued to respire in
the presence of bromacetate, although fermentation is blocked by this
substance. This contradicted the notion that respiration is necessarily an
appendix attached to a preceding anaerobic glycolysis leading to pyruvate.
The year before, Warburg (1935) had described, at the time he discovered
TPN, the oxidative transformation of G — 6 — P into 6-phosphogluconic
acid in yeast and erythrocytes, the dehydrogenase being named by him
Zwischenferment. The study of these phenomena by Warburg and by
Dickens showed that an oxidative decarboxylation with formation of a
pentose phosphate was involved. From 1950 onwards, the researches of
S. Cohen and Scott on the one hand, and of Horecker and Smyrniotis on
the other, provided new information leading to the identification of the
pentoses formed. Ribulose-5 -phosphate is first formed in the oxidation
followed by decarboxylation of the phosphogluconate brought about by
purified preparations of the dehydrogenase. Then the ribulose-5 -phosphate
is converted by phosphopentose-isomerase into an equilibrium mixture of
two pentose phosphates : ribose-P and xylulose-P.

The chain leading from the hexoses to the pentoses is theoretically rever-
sible, but this reversal is probably only of biological importance under very
special circumstances.

It is nonetheless true that in many cases a pentose phosphate can give a
hexose. But it is not by a reversal of the hexosemonophosphate oxidative
chain. The action in question is a non-oxidative action by transketolase and
transaldolase on the pentose phosphates. The demonstration of this

PRIMING REACTIONS

193

important metabolic pathway is due to Racker and to Horecker and their
co-workers. If ribose-5 -phosphate is present to act as an aldehyde acceptor,
the xylulose-5 -phosphate is rapidly converted by an enzyme in the yeast
into triose phosphate.

By means of the cycle can be explained the formation of sedoheptulose-
7-P (thus named because of its accumulation in the leaves of Sedum which
lacks the enzyme system for its further transformation).

G-6-P

2H

O = C — O

I
HCOH

I

Q't-V'dehydrogenose
TPN

HOCH
HCOH

H,0

ghtconolactonase

COOH

I

HCOH

I

HOCH

I

HCOH

— CO,

— 2H

CHjOH
C = O

HCOH
HCOH

H,COPO(OH),
S-gIuconolactone-6-P

phosphoglyconic ,

HCOH acid dehydrogenase CH,0 — PO(OH),

HsCOPO(OH),

Ru-5-P

6-phosphog!uconic acid

3-phosphogIyceraldehyde
1

Fig. 38 — Pentose Cycle
Xu = xylulose, the ketose corresponding to xylose.

The next step is the action of a transaldolase which transfers the
dihydroxyacetone group of sedoheptulose to an acceptor aldehyde
(3-phosphoglyceraldehyde) forming fructose-6-P and leaving a phos-
photetrose, D-erythrose-4-P.

A further molecule of ribulose-5-P furnishes an "active glycolaldehyde"
to the tetrose, forming a new molecule of F — 6 — P.

The overall reaction is the following :

3G— 6— P + 3O2 -^ 2F— 6— P + 1 Triose-P + 3CO2 + 3H2O

Thus in the course of the complete cycle, one molecule of glucose
is broken down to 3 molecules of COg, 3 molecules of water and a molecule
of triose ; a molecule of triose is oxidized completely.

o

194

UNITY AND DIVERSITY IN BIOCHEMISTRY

D. The Terminal Stages of Glycolysis

The glycolysis process (Embden-Meyerhof pathway) leads, as we have
said, to pyruvate. In the case of yeast fermenting glucose, the acetaldehyde
resulting from the decarboxylation of pyruvate serves as an acceptor of
electrons borne by the DPNH from the dehydrogenation of phospho-
glyceraldehyde at the time of the first oxido-reduction. Ethanol is formed.
However, the formation of acetaldehyde from pyruvic acid is not a general

CHjOH
I
CO

HOCH
I
HCOH

I
HCOH

I
HCOH
I
CH20P0(0H)j

Sedoheptulose — 7 — P

transaldolase

CH2OH
I
CO

HCOH

I
HCOH

I
CH20P0(0H)j

Ribulose-J-P

CHoOH »

I
CO

I

CH20H

Dihydroxyacetone
+

CHO

I
HCOH S9

I
HCOH
I
CH20PO(OH)2

D-erythose-4-P

CHO Diose
I
CHoOH »—

+

CHO

I

CH2OH
I
^ CO

I

HCOH

I
HCOH

I
- HCOH

I
CHaOPO(OH)2

2F-6-P

HCOH
1
CH20PO(OH)2

3 -phosphoglyceraldehyde s»

Fig. 39 (Dickens) — Formation of F — 6 — P in the hexosemonophosphate shunt.

phenomenon. It is a phenomenon peculiar to certain bacteria, yeasts and
plants which because of a particular specialization contain carboxylase. The
general phenomenon is the presence of a hydrogen acceptor more highly
oxidizing than phosphodihydroxyacetone. This oxidizing substance is
pyruvic acid in animals and in certain bacteria provided with lactic de-
hydrogenase. This enzyme in the presence of DPNH converts pyruvate
to lactate, which is the product of anaerobic carbohydrate catabolism in
animals and some bacteria. In addition, certain bacteria are specialized
to accept the same hydrogen in various other ways, either by organic
compounds such as oxalo-acetic acid, or by inorganic substances.

PRIMING REACTIONS 195

But the above are all variants operating during the anaerobic state. In the
presence of oxygen , pyruvate un dergoes the same fate in all the different organ-
is ms. It undergoes oxidative decarboxylation with productionoface^y /-Co^

Glycogen

) I glucose-6-phospliatase P

hexokinase

Glucose

ADP
ATP

G— 6— P

phosphorylate
G— 1— P

phosphogtucoisomerasc

phosphoglucomutase

phosphohexokinase
Mg"

F— 6— P

ADP\\
AT? J)

d iphosphof mctosephosphatase

F_l.6— PP

aldolase
Phosphodihydroxyacetone ^ 3-Phosphoglyceraldehyde

triosephosphate
dehydrogenase

It °™' ))h.po. )

♦ '^^ DPNH / / *

/

/

/

/

1, 3-diphosphoglyceric acid

/

/

ADP
ATP

j I phosphoglyeerate-kinaie

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

3-phosphoglyceric acid

2-phosphoglyceric acid

phosphoenolpyruic acid

phosphoglyceromutai*

enolasc

L-lactic acid

—► DPNH
DPN

" ))

N- //

lactic dehydrogenase

alcohol dehydrogenase

ADP
ATP

))

Ethanol

pyruvic acid
^

Acetaldehyde

pyruvate-kinas*

carboxylase
Mg"

Fig. 40 — Scheme to summarize reactions of glycolysis. The entry of glucose and glycogen

is shown. For other entries see Fig. 37. Two terminations are shown : that of alcoholic

fermentation and that of lactic fermentation.

196

UNITY AND DIVTERSITY IN BIOCHEMISTRY

Pyruvic decarboxylase is a complex enzyme system the coenzymes
of which are lipoic (or thioctic) acid, thiamine pyrophosphate (TPP),
coenzyme A and DPN.

Some workers maintain that a compound of thiamine pyrophosphate
and lipoic acid is present, lipothiamide pyrophosphate. The oxidative
decarboxylation of pyruvate actually occurs in three stages. In the first,
pyruvate is condensed with lipothiamide pyrophosphate (LTPP) with loss
of CO 2- The accompanying dehydrogenation results in the formation
of a thioester link (energy-rich bond). Here the dehydrogenation is not at
the start of the respiratory chain but leads to an energy-rich thioester
bond. The LTPP which has been acetylated in this first step and contains
the thioester bond reacts with the -SH group of coenzyme A an exchange
of thioacyl takes place producing a molecule of acetyl-CoA (containing
the energy rich thioester bond) and a molecule of LTPP in the sulphy-
dryl form. In a third reaction, the sulphydryl form is converted to the
disulphide form with loss of two hydrogen to DPN+ with formation of
DPNH + H+. The DPNH on entering the respiratory chain gives the
usual three ATP's.

CH3CO COOH +

LTPP

CH.CO

LTPP + CO.

HS

CH,CO ~ S

HS

LTPP + CoA — SH -> LTPP + CH3CO - S — CoA

/ /

HS HS

HS

LTPP + DPN

+

HS

/

LTPP + DPNH + H+

/

Fig. 41 — Oxidative decarboxylation of pyruvate.

II. ANOTHER PATHWAY FOR THE PRODUCTION OF
ACETYL-CoA : THE FATTY ACID CYCLE

The sulphur of CoA plays the same role for the introduction of fatty
acids into the metabolic cycle as inorganic phosphate does for molecules
of the sugars. The key to what has long been known as the Knoop

PRIMING REACTIONS 197

^-oxidation has been provided by the fatty acid cycle, the elucidation of
which owes much to the researches of Lynen. This cycle is completely
reversible and it has been reproduced m vitro, in both directions, using
purified enzymes extracted from bacteria or animal tissues.
The cycle is made up of four parts.

1. Condensation of acetyl-CoA (active acetate) with a second molecule
of acetyl-CoA to give acetoacetyl-CoA and free CoA.

CHs— CO— S— CoA .. — CH.— CO— S— CoA

HS— CoA A _CH,— CO— CHj— CO-S- CoA

2. Reduction of the acetoacetyl-CoA to j8-hydroxybutyryl-CoA

4-2H

— CHo— CO— CHo— CO— S- CoA ^

— 2H
-CHo— CH-CH2— CO— S-^ CoA

I •

OH

3. Dehydration of the /3-hydroxybutyryl-CoA to crotonyl-CoA.

— H2O
~CH2-CH— CH2— CO— S— CoA ^

I 4-H,0

OH

— CH2— CH=CH— CO— S— CoA

4. Reduction of crotonyi-CoA to butyryl-CoA

+ 2H
— CH2— CH=CH— CO— S— CoA ^

— 2H

— CH2— CH.— CH2— CO— S- CoA
FADH, ^ FAD
We started with acetyl-CoA and we have lengthened the chain by 2
carbon atoms, at the price of one acetyl-CoA molecule and four hydrogen
atoms. The breakdown of sugar can give us both of these. If we repeat the

REFERENCES

Stumpf, p. K. (1954). Glycolysis. Chemical Pathivays of Metabolism, Greenberg

(Editor) vol. 1, Academic Press, New York, 67-108.
Cohen, S. (1954). Other pathways of carbohydrate metabolism, Che?nical Pathzvays

of Metabolism, Greenberg (Editor) vol. 1. Academic Press, 173-233.
Dickens, F. (1956). The hexosemonophosphate oxidative pathway of yeast and

animal tissues. Proceedings of the 3'"'* Inter Jiational Congress of Biochemistry,

Brussels 1955, Academic Press, New York, 170-179.
HORECKER, B. L. (1958). Le cycle des pentose et sa signification physiologique.

Bull. Sac. Chim. Biol, 40, 555-578.

198 UNITY AND DIVERSITY IN BIOCHEMISTRY

operation according to the general equations above, we again lengthen the
chain by two carbon atoms. If, for example, we repeat the operation eight
times, we shall obtain stearic acid according to the equation :

9CH3— CO— S— CoA + 32H ^

^ C17H35— CO— S— CoA + 8HS— CoA + SHgO

Since the cycle is completely reversible, it also explains j8-oxidation.
Starting from a fatty acid, this acid will be activated by conversion to an
acyl-CoA derivative, it will be the object of a dehydrogenation, of a hy-
dration and a further dehydrogenation to give a ^-ketoacyl-CoA. The
thiolysis of this latter substance will give a molecule of acetyl-CoA and the
acyl-CoA of the fatty acid containing two less carbon atoms.

The overall scheme is summarized in Fig. 42.

The different enzyme reactions intervening in the fatty acid cycle are as
follows :

1. j3-ketoreductase

CH3— CH— CH2— CO— S— CoA + DPN ^ CH3— C— CHj— CO— S— CoA + DPNHj

I II

OH O

2. Ethylene-reductase

CH3— CH = CH— CO— S— CoA + FADHg ^

CH3— CHa- CH2— CO— S— CoA + FAD

3. j8-ketothiolase

R— CHa- CO— CH2— CO— S— CoA + HS— enz. ^

R— CHa- CO— S— enz. + CH3— CO— S— CoA
R— CH2— CO— S— enz. + HS— CoA ^ R— CHj- CO— S— CoA + HS— enz.

The end result being:

R— CHa- CO— CH2— CO— S— CoA + HS— CoA ^

R— CHa- CO— S— CoA h CH3— CO— S— CoA

Before entering the cycle, a fatty acid must be attached to CoA. This is
the general mechanism; it can take several forms :

1. Reaction with CoA and ATP. This is the mechanism occurring in
animal tissues. Three enzymes of differing specificity are known, reacting
with — acetate and propionate, C4 to Cjg fatty acids, and acids containing
longer chains.

2. Transfer of CoA. The transfer takes place from an acylated derivative
(for example from acetyl-CoA for the Ci to Cg acids in Clostridium
kluyveri). In the heart, and probably also in the kidney in vertebrates, such

PRIMING REACTIONS 199

a transfer takes place from succinyl-Cox\ to acetoacetate. Thus the aceto-
acetate formed in the Hver is activated, brought to the tissues by the blood
and oxidized by way of the tricarboxylic acid cycle.

III. THE TRICARBOXYLIC ACID CYCLE

Acetyl-CoA is the starting point of a series of transformations known
under the name of the tricarboxylic acid cycle. Into this cycle, at various
points, are introduced other products of the degradation of cellular
nutrients, particularly of the different amino acids. This terminal cycle,
common to the main structures forming the organism, is the chief source
of the energy-rich bonds required for biosynthesis.

Our knowledge of this cycle began with some experiments carried out by
Szent-Gyorgyi on a mince of pigeon breast muscle. This mince respires
vigorously without producing lactic acid. The respiration, at first intense,
diminishes with time. Parallel with the fall in respirator}^ activity, the
concentration of succinate in the muscle-mince decreases, but the addition
of small amounts of succinate (or of fumarate) brings about an increase in
the respiration. Since the respirator}^ quotient is equal to 1, we can conclude
that carbohydrate is being broken down. The conclusion therefore is that
the oxidation of carbohydrates is catalysed by succinate and fumarate;
succinic dehydrogenase must also play an important part because malonate
blocks the stimulant effect of succinate.

Szent-Gyorgyi having also observed the depressant effect of malonate
(an inhibitor of succinic dehydrogenase) on the respiration of the muscle
mince, he saw that the malonate must hinder the restoration bv the
succinate of the respiratory system which had become completely oxidized.

He therefore proposed the following scheme to explain respiration :

AHa . , II ». ^ reduced cytochrome >. /

H O

oxidised cytochrome ' ^ *

Succinic acid

Dehydrogenas of AH 2 Succinic Cytochrome

dehydrogenase oxidase

REFERENCES
Lynen, F. (1954). Acetylcoenzyme A and the "fatty acid cycle". Harvey Lectures,
48, 210-244.

200

UNITY AND DIVERSITY IN BIOCHEMISTRY

-CHj-CHj-CHj-CO-S-CoA
*

t
t
I

♦
-CHa— CO— S— CoA

CH,~CO-S-CoA

HS-CoA

§- ketothiolase

-CH.-CO-CHi-CO-S- CoA

elhylencrcducfaie

-CH,-CH =CH-CO-S-CoA

- HiO

H»0

-CH-CH-CHt-CO-S-CoA

(5- ketoreductase

2 H

DPN ^DPNH

Fig. 42 (after Lynen) — The enzymatic cycle for the fatty acids. (This process can also be

considered as a spiral of fatty acids, a Cg fragment being lost or gained at each turn in the

form of CH3CO — S — CoA. In Fig. 42, the change from one turn of the spiral to the next,

with addition or subtraction of a C2 fragment, is indicated by the dotted arrow.)

PRIMING REACTIONS

201

When he found that malate and oxaloacetate had the same action as
succinate and fumarate, and that their intervention is aboHshed by malon-
ate, Szent-Gyorgyi introduced the oxaloacetate-malate system into his
scheme, which became :

AH,

X

CHaCOOH

I

CO.COOH

Oxaloacetic

acid
CHj COOH

I
CH(OH)COOH

Malic
acid

COj.CGOH

I

CHXOOH

Succinic

acid

CH COOH

II

CH COOH
Fumaric
acid

ox. cytochrome

red. cytochrome

H,0

1/2 O2

dehydrogenase of
AH,

malic
dehydrogenase

succinic
dehydrogenase

cytochrome
oxidase

When it was demonstrated that the succinic-fumaric system has no
coenzyme whilst the oxaloacetic-mahc system requires the mediation of
DPN, the idea of the couphng of the two systems was recognized as
impossible and it was abandoned.

But it was known that fumarase catalyses the malate-fumarate trans-
formation, and that malic dehydrogenase catalyses the oxaloacetate-malate
conversion. From which facts sprung a new formulation of the cycle of the
dicarboxylic acids.

In the new scheme, the system catalysed by fumarase and malic dehydro-
genase is a supply system inserted laterally into the first scheme of Szent-
Gyorgyi :

malic dehydrogenase

"chTcooh

•CO COOH
oxaloacetic acid
CH2COOH
CH(OH)COOH
malic acid

DPNH

DPN

AH

fumarase i:H20

CH COOH
=^/ ^HCOOH Y '"""^'^ cytochromey y,o,
Y fumaric acid I \

A. CHsCOOH^^^j^.^^^ cytochrome^VHp
-^ ^ CH2COOH
succinic acid

-V-

"v

dehydrogenase of
AH2

succinic
dehydrogenase

Cytochrome
oxidase

202

UNITY AND DIV^ERSITY IN BIOCHEMISTRY

This particular form of the system of dicarboxylic acids had to be
abandoned when Krebs showed that a-ketoglutarate and citrate, in addition
to succinate, fumarate, malate and oxaloacetate, also re-establish the
respiration of a muscle pulp. The case of a-ketoglutaric acid, in the scheme
of Szent-Gyorgyi, did not present an insurmountable difficulty since the
oxidative decarboxylation of a-ketoglutarate yields succinic acid.

COOH
I

CH2

I
CO

COOH

a-ketoglutaric acid

+ H,0

2H

COOH

I

CHo

CH,

■f CO2

COOH

succinic acid

Apparently the entry of citric acid into the scheme can be explained by
the conversion of citrate into a-ketoglutarate under the action of a citric
dehydrogenase. But an examination of the formulas of citric acid and
a-ketoglutaric acid shows that citric dehydrogenase cannot convert the first
into the second in one step.

COOH

CH.

C(OH)COOH

I

CH2

COOH

citric acid

COOH

CHo

I
CH2

CO

COOH

a-ketoglutaric acid

It was then that the very important discovery of aconitase (Martius and
Knoop) was announced and it was demonstrated that the so-called citric
dehydrogenase was in fact a mixture of aconitase, isocitric dehydrogenase,
and oxalosuccinic decarboxylase. This multi-enzyme system explained the
passage of citrate to succinate.

COOH

PRIMING REACTIONS
COOH COOH

CH.. CHo CH,

I " itH.O I ±HoO i

C( OH) COOH ^ CHCOOH - CHCOOH

I aconitase 1| amriitmr \ isocitric |

QY\^ CH CHOH dehydrogenase CO

203

COOH

I
CH.

I
— CHCOOH ;=i

COOH
citric acid

COOH

cis — aconitic acid

COOH

isocitric acid

COOH

oxalosuccinic acid

±C02

oxalosuccinic
decarboxylase

COOH

CKj

i
CH2

[
CO

— CO2 COOH

+ I/202 I
^ CYh

oxidative '

decarboxylation ^^2

COOH
a-ketoglutaric acid

COOH

succinic acid

Following on these fundamental discoveries, Krebs formulated the cycle
in Fig. 43.

malic
4C

oxaloacetic
4C

pyruvic
3C

fu marie
4C

succinic
4C

V

7C?)

oc-ketcg'utaric -e--^/
5C f

CO2

citric CO2
6C

CO2:

Fig. 43 (Krebs) — First formulation of the tricarboxylic acid cycle.

The discovery of the mechanism for the oxidative decarboxylation of
pyruvate with formation of acetyl-CoA permitted the filling in of the miss-
ing parts of the cycle.

The cycle (Fig. 44) begins with acetyl-CoA derived from the fatty acid
cycle or from the decarboxylation of pyruvate. The entry into the cycle

204

UNITY AND DIV:ERSITY IN BIOCHEMISTRY

<
o
O

I

u

O

O X

u-u-

>-

DC

o
+

o .. « o
o o X i; o

u~u-u -u-u

O « w o

o a: a: o
u-u-u-u

O '' o
O X DC O

u-u=u-u

o

X

+1

o ^ o o

O I! DC O ■?
U-U-U-U E

6 X X

n O « O

J o x o o
g u-u-u-u

^

o

X U I

o • .. o
o O X x o
u-u -u-u-u

&0

o

o

u

o

o

o

O

i

d

<U 13

aj (u 00

u
O

CO

PRIMING REACTIONS 205

of acetyl-CoA is governed by the condensation enzyme discovered by
Ochoa. In the presence of this enzyme, acetyl-CoA reacts with a molecule
of oxaloacetate to form citric acid with the liberation of a molecule of CoA.
(If the concentration of oxaloacetic acid is low, as is the case in starvation,
diabetes, etc., two molecules of acetyl-CoA may combine to give aceto-
acetyl-CoA which, losing CoA, will give acetoacetate and eventually acetone
or other ketonic bodies). In this operation an energy-rich bond of

CH2COOH
CH3— CO— S— CoA +1 + H2O

COCOOH

oxaloacetic acid
= CH2COOH

HOCCOOH + HS— CoA

CH2COOH

citric acid

ATP is consumed. However this loss is compensated by the recovery of
an energy-rich thioester bond in acetyl-CoA. The condensation reaction
has a large —AF and is practically irreversible. Reactions 3 and 4 are
catalysed by aconitase. A molecule of water is removed in reaction 3 with
the formation of a double bond, and during reaction 4 the chain is again
saturated with the OH at another position. Reactions 3 and 4 are reversible.
Reaction 5 is a reversible oxidation utilizing TPN as the coenzyme for
the dehydrogenase (isocitric dehydrogenase). Reaction 6 is a non-oxidative
decarboxylation producing a-ketoglutaric acid. The equilibrium lies far
to the right and is practically irreversible. It is however reversible under
certain conditions. Reaction 7 is an oxidative decarboxylation having
several characteristics in common with the decarboxylation of pyruvic
acid. It can be depicted as commencing with a decarboxylation accom-
panied by a dehydrogenation, yielding a succinyl derivative of LTPP
containing an energy-rich thioester linkage. This latter, in a second
reaction, exchanges its thioester bond with CoA to give succinyl-CoA
and disulphydryl-LTPP. In a third reaction, the latter gives up two
H atoms to DPN+ giving the disulphide form, DPNH and H+. The
DPNH on entering the respiratory chain gives three ATP energy-rich
bonds.

206 UNITY AND DIVERSITY IN BIOCHEMISTRY

S

COOH— CH2— CH2— CO— COOH +

LTPP

s

COOH— CH2— CH2— CO - S

\
LTPP + CO 2

/
HS

COOH— CH2— CH2— CO - S

\

LTPP + CoA— SH ->

/
HS

HS

\
LTPP + COOH— CH2— CH2— CO - S— CoA

/
HS

HS S

\ |\

LTPP + DPN+ -> I LTPP + DPNH + H+

/ 1/

H S

Fig. 45 — Oxidative decarboxylation of a-ketoglutarate.

Succinyl-CoA, in the presence of ADP and inorganic phosphate, under-
goes an internal oxido-reduction at substrate level with the formation of
succinic acid, CoA and a molecule of ATP whose energy-rich bond arises
from the transfer of the energy of the thioester bond (reaction 8). Thus
the route from a-ketoglutarate to succinate yields four molecules of ATP.
The reaction a-ketoglutarate-succinyl-CoA reaction is reversible, but the
pathway from a-ketoglutarate to succinate is not. Reaction 9, the reversible
passage of succinate to fumarate in the presence of succinic dehydrogenase,
involves the disposal of the resulting hydrogen by a respiratory chain
differing from those to be described presently in which DPN+ and TPN+
are the initial receptors. In the case of the removal of two atoms of hydrogen
from succinic acid in the presence of succinic dehydrogenase, the electron
acceptor is cytochrome-6 followed by cytochrome-c, cytochrome-a,
cytochrome-flg and oxygen. This shortened respiratory chain gives only

PRIMING REACTIONS 207

two molecules of ATP. Reaction 10 is a reversible hydration of fumarate
to malate in the presence of fumarase. Although, in the liver, malate may-
give rise to pyruvate, this is not the case in most tissues. The malate is
dehydrogenated in the presence of malic dehydrogenase and DPN+ form-
ing oxaloacetate and DPNH. This reaction is reversible. The oxaloacetic
acid formed can, in its enolic form, react with acetyl-CoA to give citric
acid and traverse the cycle once more.

IV. RESPIRATORY CHAINS

This name is applied to the series of carriers along which pass the protons
and electrons liberated in the course of a dehydrogenation in the tricar-
boxylic acid cycle or other aerobic dehydrogenation before they reach
oxygen and unite with it to form water. As we have seen, it is along the
respiratory chain that by a series of phosphor}dations coupled to it by a still
unknown mechanism (see p. 144) the main quota of energy-rich bonds is
formed and placed at the disposition of the cells.

In the course of the successive dehydrogenations of the cycle, the
greater part of the protons and electrons liberated pass to the same series
of carriers. The first acceptor of the series being most often DPN+,
DPNH appears as the principal "fuel" in cells.

The successive transfers are the following :

Substrate + DPN+ -> oxidized substrate + DPNH + H+ (A/^ variable)

DPNH + H+ + Flavopr. -> DNN+ red Flavopr.

{AF = — 10 kcal)

red. Flavopr. + Ferricytochr. c -> Flavopr. + Ferrocytochr. c

{AF = — 16 kcal)

Ferrocytochr. c + O2 -> Ferricytochr. c (AF = — 25 kcal)

The last reaction can be split up into the following stages :

Ferrocytochr. c + Ferricytochr. a ->

Ferricytochr. c + Ferrocytochr. a

Ferrocytochr. a + Ferrocytochr. a^ ->

Ferricytochr. a + Ferrocytochr. ^3

Ferrocytochr. «3 + O -^ Ferricytochr. 03 + O

REFERENCES

Krebs, H. a. (1954). The tricarboxylic acid cycle. Chemical Pathzvays of Metabolism,
Greenberg, D. M. (Editor) vol. I, Academic Press, New York 109-171.

OcHOA, S. (1954) Enzymic mechanisms in the citric acid cycle. Advance. EnzymoL,
15, 183-270.

208

UNITY AND DIVERSITY IN BIOCHEMISTRY

succinate

t
succinic dehydrogenase

11
cy tochr. -b

ot-ketoglutaric acid
(DPT) j (CoA)

/3-hydroxybutyric acid

(dehydrogenase)

(Slater factor)

(Slater factor)

DPN
(or TPN)

Flavoprotein

I
y cy tochr. -c

\
cy tochr. -a

\
cytochr.-aj

I
Fig. 46 (Slater) — Forms of the respiratory chain.

Although DPN is the first hydrogen acceptor in the dehydrogenation
for example, of ^-hydroxybutyric acid, TPN is the primary acceptor in
other cases, and where succinic acid is concerned, the primary acceptor is
cytochrome-6.

In addition, dehydrogenations exist, such as that of a-ketoglutaric acid,
where thioctic acid acts as an intermediate between the donor and DPN.

All these paths converge at the level of cytochrome-^.

The scheme in Fig. 46 summarizes the form of the respiratory chain.

In Slater's scheme, a-ketoglutarate and ^-hydroxybutyrate are taken as
examples of hydrogen donors typical of the class to which each belongs.
The substances shown in parentheses are those which intervene at the stages
indicated, but it cannot be stated whether they give or receive protons or
electrons. This is especially the case for the Slater factor, coming between
cytochrome-6 and cytochrome-c or between flavoprotein and cytochrome-c.

In the course of the transfers just described, a series of energy-rich bonds
is formed by phosphorylations associated with the respiratory chain. In
the scheme reproduced above, a single phosphorylation has been so far
identified and this is situated at the level of the reaction between a-keto-
glutarate and thioctic acid. However the sum of experimental observations

«-ketoglutarate

succinate

succinic dehydrogenase

I
1 or 2 ~ P cyt.-6

P .. V I

— *■ thioctic y DPN > Flavoprotein y cyt.-c

acid or (Slater j

TPN factor) cyt.-a

;

cyt.-a,

I
O.

Fig. 47 (Slater) — Phosphorylations tied to the respiratory chain.

> -P

PRIMING REACTIONS

209

indicates that phosphorylations with formation of energy-rich bonds take
place in conjunction with the following links of the respiratory chain :
(1) between succinate and cytochrome-c ; (2) between cytochrome-c and
oxygen; (3) between DPN and cytochrome-c ; at this point two phos-
phorylations appear to occur.

This is summarized in Slater's scheme reproduced in Fig. 47.

substrate level

substrat
I

1

DPNH.,

oxidized substrate
(E' between 0 '
and 0.45V.)

DPN

eye. -reductase

FADH2

Fe++

■ cytochromes •

a
Fe-H-

FAD

Fe-^+ Fe++ +

J L

0.)2

0.2J V.

:\

Fe++

Fe+++

H,0

[oT]

0.0 +0.26 +0.8

oxido-reduction potential scale

i-—E; (voIm)

h

' AE equivalent to 1~P.
12 Kfoi

Fig. 48 (Lehninger) — A type of respiratory chain with a scale of oxido-reduction potentials

The sequence of the intermediates, in the case of the chain where
a-ketoglutaric acid is the model substrate (in Fig. 46) is represented with
the corresponding oxidation-reduction potentials in Fig. 48.

V. MECHANISMS FOR THE BREAKDOWN OF AMINO ACIDS

A. General Mechanisms
(a) Decarboxylation of Amino Acids

In the presence of decarboxylases amino acids give COj and an amine
according to the general reaction :

R R

I I

NH2— CH— COOH ^ NHo— CH2 -f CO2

this is referred to as "decarboxylation".

REFERENCES

Lehninger, A. L. (1955). Oxidative phosphorylation. Harvey Lectures, 49, 176-215.
Slater, E. C. (1956). Respiratory chain phosphorylation. Proceedings of the 3^'^

International Congress of Biochemistry, Brussels 1955. Academic Press, New

York, 264-277.

210

UNITY AND DIVERSITY IN BIOCHEMISTRY

The carboxylases have for their coenzyme pyridoxal phosphate which
acts according to the mechanism described on p. 174. A whole series of
decarboxylases exists, each being specific for the L-form of a given amino
acid. Certain of them have been isolated from animal tissues such as liver
and kidney, but the majority have been isolated from micro-organisms in
which the enzymes appear if their specific substrate is present in the culture
medium. In micro-organisms therefore these decarboxylases are adaptive
enzymes. The amines produced by the decarboxylation of amino acids
(Table XIII) often possess pharmacological activity; this is the case for
histamine, the product of the decarboxylation of histidine.

Table XIII

Amines resulting from the decarboxylation of various amino acids.

Amino acid

Amine

Amino acid

Amine

L-lysine

cadaverine

L-phenylalanine

phenylethylamine

L-arginine

agrnatine

L-glutamic acid

y-aminobutryic acid

L-histidine

histamine

L-aspartic acid

fL-alanine
\j3-alanine

L-omi thine

putrescine

L-tryptophan

/3-indolethyIamine

L-tyrosine

tyramine

L-cysteic acid

taurine

(b) Deaminations
1. Oxidative deamination

Many cells, and in particular those of mammalian tissues can deaminate
amino acids to form the corresponding ketonic acids in the presence of the
specific enzyme and oxygen, according to the general reaction :
R R

111
NH2— CH— COOH + — 02-^0=C— COOH-f-NHa

2

The enzymes catalysing this reaction are the L-amino acid oxidases and the
D-amino acid oxidases. The role of the latter in metabolism has not yet been
elucidated, for the naturally occurring amino acids are generally of the L-series.

The D-amino acid oxidase of sheep kidney has been purified; it is a
flavoprotein containing FAD. It is of low specificity and catalyses the oxida-
tive deamination of all the amino acids of the D-series with the exception of
glutamic acid. It does not act on amino acids of the L-series, or on glycine.

The L-amino acid oxidases isolated from various animal cells and micro-
organisms are also flavoproteins, containing FMN, but they likewise
are not very specific; they act on a number of amino acids, but not on all.
Glycine is deaminated neither by D-amino acid oxidases nor by L-amino
acid oxidases. Its oxidative deamination is accomplished in the presence
of a specific enzyme, glycine-oxidase, in the following manner :

NH2CH2COOH + iOa -> NH3 + CHO— COOH

glyoxylic acid

PRIMING REACTIONS

211

In the presence of the same enzyme, methylglycine or sarcosine is
degraded as follows :

CH3NHCH2COOH + iO, -^ CH3NH2 + CHO— COOH

methylamine glyoxylic acid

Another specific enzyme of oxidative deamination is glutamic acid
dehydrogenase. This is an anaerobic dehydrogenase:

L-glutamic acid

Imino acid of
glutamic acid

DPN

TPN

DPNH2
TPNH

L-glutamic acid
dehydrogenase

2H-

respiratory chain

a — ^ketoglutaric acid

spontaneous reaction
^NHa

FAD can utilize molecular oxygen as an electron acceptor and hydrogen
peroxide is formed. The oxidative deamination of an amino acid in the
presence of an amino acid oxidase containing FAD can be written as
follows:

R R

NH2— CH— COOH + F AD->N H=C-COOH + F ADH^

R R

NH=C— COOH + H20-*0=C— COOH + NH,

FADHo+02-^FAD+H,02

catalase

H2O

2^^2

->HoO+— O2
2

R

R

NH2-CH-COOH4- — 02-^0=C-COOH+NH3

2

212 UNITY AND DIVERSITY IN BIOCHEMISTRY

In the absence of catalase, the hydrogen peroxide oxidizes the ketonic
acid with formation of the aUphatic acid having one carbon atom less and
the overall reaction becomes

R R

NH2— CH— COOH+Oo-^COOH+COj+NH,

2. Non-oxidative deaminations

Enzymes also exist, in the case of serine, threonine and homoserine,
which can catalyse a non-oxidative deamination commencing with a
dehydration of the substrate.

In the case of serine for example, the mechanism is as follows :

— H2O
HOCH2CHCOOH > CH2=CCOOH<=^CH3C-COOH

^^^ serine dehydrase ^^^ ^^

serine a — aminoacrylic acid a — iminopropionic acid

H2O

CH3CCOOH >CH,COCOOH + NH3

II

NH hydrolysis

a — iminopropionic acid

{c) Deamidations

The deamidation of glutamine and asparagine have already been con-
sidered with the enzymes catalysing the reaction (p. 156).

{d) Decarboxylation of the Ketonic Acids formed by Deamination

of Amino Acids

These reactions are brought about by four types of decarboxylases.

1. a-ketodecarboxylases

The a-ketonic acids are decarboxylated in the presence of these enzymes
with formation of the aldehyde having one carbon atom less and liberation
of CO2. The coenzyme is DPT. The carboxylase is formed by the union of
the coenzyme and the specific protein. This protein appears to be present
only in plants and micro-organisms, whilst DPT is present in animal
tissues also. The decarboxylation of pyruvic acid to form acetaldehyde
during alcoholic fermentation is due to an a-ketodecarboxylase.

2. Oxidative a-ketodecarboxylases

One example has already been described at the point of entry of pyruvic
acid into the tricarboxylic acid cycle.

PRIMING REACTIONS

213

3. ^-ketodecarboxylases

An example of this type of decarboxylation is the action of oxaloacetic
decarboxylase which is present in animal tissues and in many micro-
organisms.

COOH COOH

c=o

I
CH.,

I
CH,

4- CO,

COOH

4. Oxidative ^-decarboxylases

The oxidation of a ^-hydroxyacid with decarboxylation of the j8-ketonic
acid formed has already been described above in the case of the passage
of isocitric acid into oxalosuccinic acid and then to a-ketoglutaric acid
during the tricarboxylic acid cycle.

Another enzyme of the same type has been discovered by Ochoa in
animal tissues : it is known as "malic enzyme" and catalyses the oxidation
of malic acid to pyruvic acid and COg.

COOH

TPN+ + CHOH ^ TPNH +

CH^

I
COOH

malic acid

COOH

I

c=o

CH,

COOH

oxaloacetic acid

COOH

I
^ C=0 + CO,

Mn+ +

CH,

pyruvic acid

The equilibrium constant favours the reaction occurring from left to
right but the reverse reaction occurs if the TPN formed is continuously
removed. The oxaloacetic acid is shown in brackets because it does not
appear in the free form during the reaction. Under the action of the enzyme
it is decarboxylated straight away; the enzyme must not be confused with
malic dehydrogenase which requires DPN as its coenzyme.

{e) Tracts aminations

All the naturally occurring amino acids can in vivo participate in trans-
amination reactions catalysed by tra?isaminases. The reactions are universal
in the biosphere in which they play an important role. They form important
metabolic links between aspartate, glutamate and alanine on the one hand,
and their corresponding a-keto acids in the tricarboxylic acid cycle on the

214 UNITY AND DIVERSITY IN BIOCHEMISTRY

Other, The most active and the most widely distributed transaminase is
the glutamic-oxaloacetic enzyme :

L-glutamic acid + oxaloacetic acid ^

a-ketoglutaric acid + aspartic acid

The following reaction is also very common :

amino acid + a-ketoglutaric acid ^

a-ketonic acid + glutamic acid

The participation of oxaloacetic acid in transaminations appears to be
limited to the glutamic-oxaloacetic system. Another common system is the
glutamic-pyruvic one :

L-glutamic acid + pyruvic acid ^

a-ketoglutaric acid + L-alanine

It has long been thought from our knowledge of these two systems that
one of the members of the pair of substrates for a transaminase must be a
dicarboxylic acid. Since then, leucine-pyruvate, phenylalanine-pyruvate
and ornithine-pyruvate transaminations have been demonstrated. However
it is not possible to exclude the presence of a trace of glutamate, thus :

pyruvic acid + glutamic acid ^ alanine + a-ketoglutatic acid
amino acid + a-ketoglutaric acid ^ a-ketonic acid + glutamic acid

Numerous transaminases exist. Their specificity appears to be narrow in
some cases and very much wider in others.

Each transaminase consists of a specific apoenzyme and a coenzyme
which is pyridoxal phosphate.

It was long believed that glutamine and asparagine did not take part in
transamination reactions except after hydrolysis. In fact enzymatic systems
have been demonstrated which catalyse transminations from glutamine and
asparagine to many ketonic acids. Glutamine is an even better donor than
glutamate but the specificity of glutamine transaminase for the a-ketonic
acid is low.

(/) Trans deaminations

One of the mechanisms which has been proposed to account for the
oxidative deamination of amino acids invokes a transamination followed by
a deamination. This mechanism appears to be capable of explaining the
rapid and reversible deaminations whose character is not in accordance
with the properties and action of the L-amino acid oxidases. A transami-
nation to a-ketoglutaric acid would remove the amino group from an

PRIMING REACTIONS

215

amino acid and the glutamic acid formed would be deaminated by the
specific L-glutaminase. In addition this mechanism accounts for the rapid
synthesis of amino acids from ammonia and a-keto acids.

NH2 — CH-COOH a-ketoglutarate

R

0=C— COOH

L- glut a mate

NH3-h 2H

glutamic
dehydrogenase

B. The Complete Degradation of various Amino Acids

[a] Glutamic Acid

As we have just seen, L-glutamic acid is not deaminated by the action of
the L-amino acid oxidase of animal tissues and bacteria. But, in the presence
of a specific enzyme, glutamic dehydrogenase, it undergoes oxidative
deamination in the presence of either DPN or TPN. This reversible re-
action gives a-iminoglutaric acid.

COOH COOH

CH.

CH,

CH,

+ DPN+

CH2

4- DPNH + H+

NH2— CH— COOH NH=C— COOH

L — glutamic acid a — iminoglutaric acid

The a-iminoglutaric acid is hydrolysed spontaneously to a-ketoglutaric
acid and ammonia. The a-ketoglutaric acid can enter the tricarboxylic acid
cycle.

(b) Aspartic Acid

Aspartic acid, by transamination, gives oxaloacetic acid which then also
enters the tricarboxylic acid cycle.

(c) Histidine

Histidine, besides its connection with the metabolism of pentoses,
nucleotides and certain other amino acids, follows a path leading to
glutamic acid. In certain bacteria, the enzyme system forms one mole of
glutamic acid, one mole of formic acid, and two moles of ammonia from one
mole of histidine, whilst in other bacteria one mole of glutamic acid, one

216

UNITY AND DIVERSITY IN BIOCHEMISTRY

mole of formamide and one mole of ammonia are produced. The separate
steps of this degradation have been worked out from a study of a number of
systems and are shown in Fig. 49 (still hypothetical steps are between
brackets).

H

N NH

I NH,

HC=C — CH,— CH— GOGH

1 histidase, histidine deaminase
H

N NH

II + NH, + H,0
HC=C — CH = CH — COOH^- --?"-
urocanic acid

I

H|

^\

N NH

I

I

o=c

CH— CH,— CHj— COOH J

H

^\

N NH
.HO — CH— C= CHI— CH, — COOH.

Hj

/%

N NH

HO— C == C— CH,— CH,— COOH .

imidazole propionic acid

I

I
NH

II
CH

NH

CHO

NH

+ NH,

I + H,0 I
HOOC— CH— CH,— CH,— COOH > HOOC— CH— CH,— CH,— COOH

formamidinoglutaric acid formylglutamic acid

I + H,0 I + H.O

i I

O NH,

HC + HOOC— CH— CH,— CH,- COOH

NH,

formamide

NH,

HOOC— CH—CH,—CH,— COOH + HCOOH
glutamic acid formic acid

glutamic acid

Fig. 49 (after Tabor) — Degradation of histidine.

The glutamic acid can enter the tricarboxylic acid cycle through a-keto-
glutaric acid, so histidine is thus degraded to COg and water.

(d) Leucine, Isoleucine, Valine

The degradation of leucine, isoleucine and valine operates by oxidative
deamination and then decarboxylation of the corresponding keto-acids.

Leucine is first transformed into a-ketoisocaproic acid. This latter
combines with CoA and is oxidized to senecioyl-CoA, this is followed by a
hydration with formation of j8-hydroxyisovaleryl-CoA. Then, in the course
of a reaction requiring ATP, COg is attached to the end of the chain and the
^-hydroxy-^-methylglutaryl-CoA formed is split into acetoacetic acid

PRIMING REACTIONS

217

CHs CH3

\/
CH

CH3 CHs CH3CH3 CH3 CH3 CH3 CH2

CHa-

I

C:
I

s

CH CH

CoA I — CO2 I

CH2-< C = O-

O

C = O

I
COOH

COOH

CH

I

-*-c = o

I

s

I

CoA

CH

O

+ CoA

1 — COa
COOH

CH3 CH3 CH2

\/
CH

I

c = o

I

s

I

CoA

CH3

o

CoA

isovalers'l-
CoA'

Ik

CH3 CHs

\/
C

II
CH

I

c = o

I

s

a-ketoiso- a-ketoiso- isobutyryl-
caproic acid valeric acid CoA

a-keto-

j3-methyl,valeric

acid

L-methyl-butyryl-

i CoA

CHs CH3

\/
CH

I
CH2

I
CHNH2

I
COOH

CH3 CHs CHs CH2 CHs CHa — CHs CHs

CH2

CH3

CH

I
CHNH2

I
COOH

\aline

CoA

senecioyl-CoA Leucine

CHs CHs

\/
COH

I
CH2

I

c = o

I

s

I

CoA

^-hydroxv-iso\alervl-CoA

I

COOH

I
CH2

I
CH3COH •

I
CH2

I

c = o

CoA
^-hydroxy-;S-methyI-glutary-CoA

r

CHs CHs

I I

c = o c = o

I I

CH2 + s

I I

COOH CoA

acetoacetic acetyl-
acid CoA

C

I

c =

I

s

I

CoA

O

CH

I
CHNH2

I
COOH

Methacryl-CoA Isoleucine

CHs CH2OH

\/
CH

c = o

S

I
CoA

C

I

c = o

I

s

I

CoA

tiglyl-CoA

' + H2O
>|
CHs CHOH — CHs

\/
CH

1

c = o

I

s

I

CoA

a-methyl-j3-hydroxy-
butyryl-CoA

i^DPN +
VDPNH

CHs CO — CH3

\/
CH

c = o

s

I

CoA
a-methyl-acetoacetyl-CoA

+ CoA

CHs

1

>

CH3

1

1
C =

1

0

CH2

1

1

s

1

c! = o

1

CoA

1
S

1
CoA

acetyl-CoA

P

ropionyl-CoA

Fig. 50 (after Coon) — Degradation of leucine, valine and isoleucine.

218

UNITY AND DIVERSITY IN BIOCHEMISTRY

CHaNH2

I
(CH2)s

I

CHNHj
I
COOH

®i 11®

CHjNHAc
I

(CH2)9

CHNHj
I
COOH

®\ 1©

Lv<inf

Nj=z Neurospora
k)= Rat

COOH

®1|®

CHO

I
(CHj),

I
CHNHAc

I
COOH

.@.

© ':°°" Qy

COOH

• (CH,)3 -

0 L CN

N-acetyl a-aminoadipic-
e-semialdehyde

CHNHj

COOH

a — aminoadipic acid

(CH2)a
I

c = o

I
COOH

■* glutaric acid

®

a— ketogluttric
acid

Fig. 51 (after Work) — Degradation of lysine.

PRIMING REACTIONS

219

and acetyl-CoA. Isoleucine undergoes a similar fate, the fixation of COg
being replaced by a dehydrogenation. Finally, acetyl-CoA and propionyl-
CoA (Fig. 50) are obtained.

The path of valine joins that of leucine at the level of a-ketoisocaproic
acid.

NH,
1
CHjCH-COOH

hydroxyl«»e ipK-

f
CHjCH-COOH

O

phenylalanine

Fe++
O

CHjC-COOH

OH

O

II

V

OH

tj'rosine

stcorbste

V

+ 0

— COa
+ 0

OH OH

p-hydroxyl-phenylpyruvic acid 2,5-dihydroxy phenylpyruvic acid

(1^^=^ CHjC-COOH

u

OH

COOH

CH5COOH H

+ o»

/

Fe-H-
~SH H

H,

Ha

H H H , „^
HOOC ^C ,C ,C +"»"
GSH V^ \^<^ \^^ \

OH

homogentisic acid

^ /C ^<-

^C ^C XOOH

II

o

II
o

maleylacetoacetic acid

H

C
OH

C
OH

COOH

fumarylflcetoacetic acid

HOOC

I
HC
II
CH

I

COOH
fumaric acid

O

II
+ CHj-C-CHjCOOH

acetoacetic acid

Fig. 52 (after Knox) — Degradation of phenylalanine and tyrosine.

(e) Lysine

Lysine does not participate in the general deamination reactions of the
amino acids. It is degraded by way of pipecolic acid both in animal tissues
and in plants. The steps leading from lysine to a-ketoglutaric acid shown
in Fig. 51, have been demonstrated both in the rat and in the mould
Neurospora.

As the scheme shows, the various steps of lysine catabolism are not all
reversible and though they have all been demonstrated to occur in the rat
this is not the case for Neurospora.

(/) Phenylalanine and Tyrosine

Phenylalanine and tyrosine are degraded as far as fumaric acid and
acetoacetic acid as shown in Fig. 52.

220

UNITY AND DIVERSITY IN BIOCHEMISTRY

J

NH,
I (bacteria)

•CHs-CH-COOH ^

pyridoxalphosphate

tryptophan

O

II

NH,

-C-CH,.^CH-COOH

-N-CH
H M
O

(orinylkymirenin

/\

V

O NH2

II I

— C-CH,-CH-COOH

kvnurcnin

0

-C-CHa-CH— COOH

HO-CH2-CH-COOH +
I
NHj

serine

Af

V^

N

/

O

II
CHj-C-COOH

indolepyruvic acid

<^

COOH

NH2

NH2
OH
]-hydroxykynurenin

0

anthranilic acid

OH

V^N

^COOH

COOH

NH2

kynurenic acid

OH

WAj^^COOH
OH

xanthurenic Bcid

COOH

OH

3 -hydroxy anthranilic acid

^^

quinolinic acid

indole

/

COOH
NH.

anthranilic acid

CH,-CH-COOH

I
NH,.

itaninc

COOH

Z'

N
oicotinic acid

Fig. 53 — Degradation of tryptophan.

PRIMING REACTIONS 221

is) Tryptophan

The ketonic acid corresponding to tryptophan is a-keto-^-indolyl-
pyruvic acid, which can be obtained by transamination. The metabohsm
of this substance is unknown but it is not the main metaboUc pathway for
tryptophan which follows a series of different paths, as shown in Fig. 53.
The most notable of these is the one leading to nicotinic acid.

The main pathway of tryptophan breakdown is still unknown.

(h) Glycine and Serine

Glycine can follow any one of a number of paths and during metabolism
it may be transformed into a variety of substances : formate, acetate,
ethanolamine, serine, aspartic acid, fatty acids, purines, pyrimidines,
ribose or protoporphyrin. Its complete degradation, like that of serine or
ethanolamine into which it is readily transformed, may be brought about by
conversion to pyruvic acid from whence it can enter the glycolysis chain
or the tricarboxylic acid cycle.

CHoOH CH2OH CH2 CH, CH3

I " I II I I

CH0NH2 CHNH, - H.O C-NH2 C=NH + H2O CO + NH,

^1 - I -I - I

H-COj COOH COOH COOH COOH

serine pyruvic acid

But this is not the only entry of glycine into the priming reactions.
Glycine can be converted into COg and water by means of the Shemin
cycle, where the catalyst is not oxaloacetate as in the Krebs cycle, but
succinate, whose active form, succinyl-CoA, condenses with the glycine.
The Shemin cycle can therefore be worked into that of Krebs to form a
shunt (Fig. 54).

The succinyl-CoA condenses with glycine, at the a-carbon atom, to
form a-amino-^-ketoadipic acid which is then decarboxylated to 8-amino-
levulinic acid. The latter is deaminated to ketoglutaraldehyde which is
oxidized to ketoglutaric acid. This compound enters the tricarboxylic acid
cycle or is decarboxylated to succinic acid. In one revolution of the cycle
one molecule of glycine is completely oxidized to CO2 and water.

(/) Proline

Proline is first oxidized to glutamic acid whose metabolic path it then
follows. Hydroxyproline gives rise to ;8-hydroxyglutamate-semialdehyde.

222

UNITY AND DIVERSITY IN BIOCHEMISTRY

Ci fragments (formaldehyde or
formate) from C-a of glycine
and used for the synthesis of
purines, etc.

a — ketoglutaric -<-
acid

Ac.

uccmyl — CoA
+ glycine

H

HOOC— CH,— CH,— C— CHO

&

ketoglutaraldehyde

HOOC— CH,— CHs— C— C— COOH

II I
ONHj
a — amino — P — ketoadipic acid

—CO,

HOOC— CH,— CH,— C— CH,— CH, NH,

II
O

0 — aminolevulinic acid

— NH, •"

+ }0,

porphyrins

Fig. 54 (Shemin) — The succinate-glycine cycle.

(j) Cystine and Cysteine
Among various other metabolic fates these two acids can yield pyruvic acid.

HOOC— HC— HjC— S— S— CH,— CH-COOH

I cystine I

NHa NH2

Ha i T Oj

HS— CHa— CH— COOH -* HjS + NH., + CH,— C— COOH

I desulphydrase

NHa O

cysteine pyruvic acid

Quantitatively the most important path is the following :

SH SO2H

CH,

CHNH2

CH2
CHNH2
COOH COOH

cysteine cysteine-sulphinic acid

Cysteine-sulphinic acid, by transamination, can yield j8-sulphinyl-
pyruvic acid which is decomposed into pyruvic acid and SO, — which

PRIMING REACTIONS 223

oxidizes to SO4 — . The cysteine-sulphinic acid can also be oxidized to
cysteic acid, or decarboxylated to form taurine.

(k) Alanine

It is deaminated to pyruvic acid which enters the cycles of priming
reactions.

C. The General Scheme

The overall plan in Fig. 55 shows the general pathways for the complete
oxidation of the amino acids. This does not necessarily mean that the
amino acids always follow these paths. There may exist, and do exist,
particularly in animals, numerous interrelations between the metabolic
paths of the amino acids. Figure 55 shows the most important ways in
which the carbon chains of the amino acids can be oxidized completely.

VI. INTERRELATIONS BETWEEN PRIMING REACTIONS

These are represented as shown in Fig. 56. Certain aspects, such as the
relation between pyruvate and oxaloacetate, or malate, will be explained later.

Figure 56 shows the interrelations between glycolysis and the hexo-
semonophosphate shunt at the level of G — 6 — P and F — 6 — P, between
glycolysis, the fatty acid cycle and the tricarboxylic acid cycle at the level
of acetyl-CoA, between the tricarboxylic acid cycle and the succinate-
glycine cycle at the level of succinyl-CoA.

Figure 56 also shows the points of entry of the various amino acids into
the series of priming reactions.

VII. ENERGETICS OF THE PRIMING REACTIONS

A. Glycolysis

In Fig. 57 are shown the values of —AFq in kilocalories per mole for the
conversion of glucose into alcohol or into lactic acid and for the conversion
of glycogen into lactic acid. A part of this free energy is lost as heat but the
remainder is retained in reserve in the form of ATP energy-rich bonds.
A balance-sheet can be drawn up for the free energy and for the phosphate
bonds and from it we can determine the efficiency of the process. In the
course of the phosphorylation of glucose and of the phosphorylation of
F — 6 — P, in each case a mole of ATP has been used up. On the other hand,

REFERENCES

Meister, a. (1955), Transamination, Advanc. Enzymol. 16, 185-246.

McElroy, W. D. and Bentley Glass H. (Editors), 1955.^ Symposium on Amino
Acid Metabolism. Johns Hopkins Press, Baltimore.

Fromageot, C. (1955), The metabolism of sulfur and its relations to general meta-
bolism. Harvey Lectures 49, 1-36.

224

UNITY AND DIVERSITY IN BIOCHEMISTRY

PRIMING REACTIONS 225

for each mole of triose which has undergone oxido-reduction two moles of
ATP have been formed, making four moles per mole of glucose. The net
gain is therefore two moles of ATP per mole of glucose.

The free energy liberated when a mole of glucose is converted into two
moles of lactate is 49,700 calories. Two energy-rich bonds of ATP have
been recovered representing 24,000 calories, so the overall yield is one
of 47%.

In the case of glycogen the formation of G — 1 — P in the presence of
phosphorylase and inorganic phosphate takes place without the interven-
tion of ATP (see Fig. 38). Consequently, only one molecule of ATP is used
up in the phosphorylations at the beginning of the chain. Hence, the net
gain per mole of glycogen monomer is three moles of ATP.

Since the free energy liberated in the course of the passage of the
monomer to lactic acid is 56,700 calories and 36,000 calories is regained,
the yield is 63%.

B. The Tricarboxylic Acid Cycle

The changes in free energy which accompany the various stages of the
cycle are shown in Fig. 56.

When we estimate the energy yield it must be noted that the molecules
of pyruvic acid which enter the cycle by way of acetyl- Co A come from the
glycolysis chain, but that the DPNH formed during the oxidation of
phosphoglyceraldehyde has not been taken into account. Since each
dehydrogenation involving DPN or TPN yields three moles of ATP
per mole of substrate, when glucose has provided two moles of pyruvate,
8 moles of ATP have been formed at the same time. In the tricarboxylic
acid cycle we have a series of dehydrogenations where the primary acceptor
is DPN. They are the malate-oxaloacetate and a-ketoglutarate-succinyl-
CoA reactions. Succinyl-CoA when it decomposes gives a further mole
of ATP. The succinate-fumarate reaction does not require the media-
tion of DPN and only two energy-rich bonds are formed. Thus during
one complete turn of the cycle, that is for all the various stages from
citrate to oxaloacetate, twelve energy-rich bonds of ATP are obtained :

isocitrate -^ oxalosuccinate 3

a-ketoglutarate -^ succinate 4

succinate -> fumarate 2

malate -> oxaloacetate 3

12

When pyruvate is condensed with oxaloacetate to form citrate, three
moles of ATP are formed per mole of pyruvate, or six per mole of glucose.

226

UNITY AND DIVERSITY IN BIOCHEMISTRY

amino
•cid«

glucow pho8phogluconic

C-6-P

/

i[

I

pentosephosphates

F-«-P

P l,< PP 5edoh«ptulose-7-P

trioicphosphates

3-phoaphogIyceric add

{{

-CHa-CH = CH-CO-S-CoA

-CHj-CHj— CH,— CO— S- CoA

-CH2-C-CH,-CO-S- CoA
OH

■//

-CHj-C-CHj-CO-S-CoA

HSCoA

acccoicetylCoA

osuccinate

acccoacctaie

amino actda

fumarate

ketoglutaraldehyde
+ glycine

a — ketoglutarate * ' amino acids

succinylCoA

luccinate ' +

acetoacetyl CoA

Fig. 56 — Interrelation of the various priming reactions.

glycogen

PRIMING REACTIONS
glucose

227

-56-7

lactate

-49-7/
/

2 lactates *^

\-62-2

- 45-8 I - 45-8
alanine > pyruvate

CO,

ethanol

I acetate

i - 46-5 /

acetaldehyde / +7-4

- 55-0 I /

I _ 48 ^ >/ + 6-S
acetylcoA

+ oxaloacetate
- 7-8

citrate
I +2-04

cis-aconitate
1 -0-45

isocitrate
I -47-0

oxalosuccinate
I - 8-6

- 45-9 i

glutamate >■ a-ketoglutarate + COg

I - 69-8

succinate + COg
I - 35-7

I
fumarate
I - 0-88

malate
I - 44-8

- 44-7 j
aspartate >■ oxaloacetate

2 ethanols + CO,

1 /2 butyrate

i -43
1 /2 acetoacetate

Fig. 57 (Krebs) — Changes in free energy (^Fo) in kcal. per mole at 25°, pH 7-2, 0-2 atm.
0,, 005 atm. COj. and the other reactants having a concentration of OOIM. The free
energy changes include changes due to associated reactions notably reactions with
molecular oxygen acting as an acceptor for hydrogen removed at various stages.

Consequently, in the overall reaction for the oxidation of glucose, the moles
of ATP formed from ADP add up as follows :

glucose —*2 pyruvate -{- 2 HoO 8

2 pyruvate + 2 oxaloacetate + Oj -^ 2 citrate + 2 CO2 6

2 citrate + 4 O2 -* 2 oxaloacetate + 4 HjO + 4 CO2 24

Total 38

The overall reaction can thus be written

CeHiaOe + 6O2 + 38ADP + 38P -> 6H2O + 6CO2 + 38ATP
If glycogen is the starting point, we may add 1 ATP, giving a total of
39 ATP.

228 UNITY AND DIVERSITY IN BIOCHEMISTRY

C. The Fatty Acid Cycle

Here, the calculation of the energy yield is less certain. It is believed that
5 energy-rich bonds are formed as each acetyl group is split off. Now each
of the latter as it traverses the tricarboxylic acid cycle will give twelve
energy-rich bonds (one complete turn of the cycle).

One mole of palmitic acid gives eight acetyls, so that the production of
ATP during the passage of a mole of palmitic acid through the priming
reactions will be as follows :

palmitic acid + 10^ -> 8 acetyls 7 X 5 = 35

8 acetyls + 16 Og -> I6H2O + I6CO2 8 x 12 = 96

Total 131

The overall reaction can be written :

C16H32O2 23O2 + 131ADP + 131? -^ I6H2O + 16COa + 131ATP

D. Degradation of Amino Acids

Let us take the example of alanine, the heat of combustion of which is
392,000 cal per mole: on deamination it will yield a mole of pyruvate. The
latter will be oxidized by the respiratory mechanism yielding energy-rich
bonds calculated as follows :

pyruvate + oxaloacetate + O -> citrate+C02 3

citrate + 23 O2 -> oxaloacetate + 2 H2O + 2 CO2 12

15

E. Remarks on the Preceding Calculations

These calculations must be accepted with caution. In fact they imply
values which may in some cases only be approached rather than attained.
For example the P/0 ratio in the course of oxidative phosphorylations.
Moreover they assume a perfect coupling between phosphorylations and
oxido-reductions. In practice numerous factors bring about the uncoupling
of these two processes, examples are dinitrophenol and thyroxine. There
are good reasons to believe that even in a given species the efficiency of
coupling varies from one individual to another.

Furthermore, the values used in the above calculations have been
obtained from measurements made in vitro on systems of the purified
substances. This is not the situation in the cell. Most important is the fact
that it contains an enzyme, adenosine triphosphatase, which hydrolyses
ATP into ADP and inorganic phosphate, and it is not impossible that in
vivo part of the ATP formed is lost through the action of this enzyme, also
this enzyme is involved in a specific way in the transformation of ATP
bond energy into work.

CHAPTER III

BIOSYNTHESES

I. THE MATERIALS FOR BIOSYNTHESES

The priming reactions described in the preceding chapter are the principal
sources of the coin which pays for the performance of cellular work : the
pyrophosphate bond of ATP. This coin also pays for the energy required
in biosyntheses, with the exception of assimilation phenomena whose
mechanism will be studied later (see Part Six). But the priming reactions do
not furnish only ATP, they also provide a series of construction materials
for these biosyntheses. For example two-carbon fragments in the form of
"active acetate" or acetyl-CoA are obtained during the priming reactions
from glucose, fatty acids, acetoacetic acid and amino acids.

There are also one-carbon atom materials, together with CO2 which is
liberated at numerous points in the priming system. But the most important
material of this type is that referred to as "formate" (Ci) that is CHO-
or active formyl, into which can be converted not only formic acid, formal-
dehyde and methanol, but also the a-carbon of glycine, the a-carbon of
glycollic acid, and a- and ^-carbons of serine, the a-carbon of threonine,
the C — 2 of histidine, the C — 2 of tryptophan, the a-C and the C — 6 (or
C — 2) of phenylalanine and tyrosine, etc. Acetone can be split into an
acetyl fragment and a formyl fragment.

CH9

H— C==0 Formyl

c=o -* +

CH3 C=0 Acetyl

Acetone |

CH3
In certain bacteria (but not in mammals), pyruvic acid can undergo
fission into acetyl phosphate and formic acid (phosphoroclastic reaction),
providing Ci units.

CH3

I
C=0 CH3

I I

COOH +H3P04^ 0=C— O— PO,H, H- HCOOH

pyruvic acid acetyl phosphate formic acid

229

230 UNITY AND DIVERSITY IN BIOCHEMISTRY

One-carbon materials also exist which are not in the oxidized form as in
CO2 and Ci, but are in the reduced form of labile methyl groups (CH3-).
These labile methyl groups can give Ci units and they originate from Ci
units.

By oxidation it is possible to pass from Ci to COg, but the reverse reaction
is impossible, at least in mammals.

Glycine is another material which is used. These molecules of glycine
come from the proteins of the diet, from the degradation of serine or
threonine, or are provided by synthesis from pyruvic acid.

In addition, biosyntheses draw materials of a more complex structure
from different points in the priming reactions, and in particular from the
tricarboxylic acid cycle.

II. BIOSYNTHESIS

A. Biosynthesis of Simple Chemical Structures

(a) The Utilization of CO 2 to Form — C — C — Bonds {excepting

Assimilation by Autotrophes)

This is essentially the opposite of the reversible decarboxylations (p. 212)
of ketonic acids. We shall therefore consider jS-carboxylation, the inverse
of the decarboxylation catalysed by a j8-ketodecarboxylase, and a-carboxy-
lation, the inverse of the decarboxylation catalysed by an a-ketodecarboxy-
lase (see pp. 212 and 213).

1. fi-carboxylation

This process for the fixation of CO2 is very important. Two examples
are, fixation by pyruvic acid to form oxaloacetic acid, and fixation of CO2
by a-ketoglutaric acid. The former is the mechanism for the synthesis of
dicarboxylic acids and the latter for the synthesis of tricarboxylic acids.

{a) Fixation of CO2 by oc-ketoglutaric acid. — The conversion of isocitric
acid into a-ketoglutaric acid and COg in the tricarboxylic acid cycle has
been shown by Ochoa to take place in two stages :

isocitric acid + TPN+ ^ oxalosuccinic acid + TPNH (1)

Mn++

oxalosuccinic acid ^ a-ketoglutaric acid + CO2 (2)

The sum of (1) and (2) is

Mn++
isocitric acid + TPN+ ^ a-ketoglutaric acid + COg + TPNH (3)

BIOSYNTHESIS 231

Reaction (3) can be caused by proceeding from right to left by coupling
the system to an oxido-reduction system capable of reducing TPN, for
example the glucose-6-phosphate dehydrogenase system, and we obtain

G— 6— P + TPN+ ^ 6-phosphogluconic acid + TPNH (4)

a-ketoglutaric acid + COj -f TPNH ^ isocitric acid + TPN+ (5)

The sum of (4) and (5) gives

G — 6 — P + a-ketoglutaric acid + COj ^ 6-phosphogluconic acid

+ isocitric acid (6)

If aconitase is added, the equilibrium is shifted still more in favour of
COj fixation since 90% of the isocitric acid is continuously removed.
Since the enzyme systems here described are universally distributed in
cells, both in animals and in plants and in micro-organisms, this means of
fixation is an important one.

(b) Fixation of CO 2 by pyruvic acid. — The entry of pyruvic acid into
the tricarboxylic acid cycle by way of acetyl-CoA, then by formation of
citric acid, depends upon there being a sufficiently high concentration of
oxaloacetic acid. This concentration is maintained by the fixation of CO,
by pyruvate with formation of oxaloacetate.

From the moment that the C4 acids are used up, COg becomes essential
for the continuation of respiration by means of the cycle.

The discovery of the fixation of COg by heterotrophes was first made in
the propionic acid bacteria (Wood and Werkmann). In these bacteria when
fermenting glycerol the CO2 is utilized for the formation of succinic acid.
Elsden has shown that for E. coli the rate of formation of succinate in
the presence of pyruvate depends upon the CO2 pressure.

The CO2 is incorporated into succinate according to the following
reactions :

CHs +CO2 COOH +H2 GOGH -H2O COOH -fHa GOGH

► I > I > I > I

CO CH2 GH2 GH CH,

I I t II I

GOOH CO GHOH GH CH2

1 i I I

COOH COOH COOH COOH

pyruvic acid oxaloacetic acid malic acid fumaric acid succinic acid

y

Wood-Werkmann Reaction

By use of labelled carbon it has been shown that COg fixed by pyruvate
contributes to carbohydrate synthesis in heterotrophes. For example, the

232

UNITY AND DIVERSITY IN BIOCHKMISTRY

liver glycogen of animals contains carbon atoms derived from COj,
(those which occupy the 3 and 4 positions in the hexose molecule).

This proves that glycolysis is partially reversible, a COg molecule being
introduced into a triosephosphate and passing through 3-phosphoglyceric
acid to glycogen.

I

CH2OPO3H2

I
CHOH

I
*COOH

*COOH

CHOH

CH2OPO3H,

3-phosphoglyceric acid

CHaOPO.H,

CHOH
'^CHO
" CH.OH

CO

CH20P03H2

Triosephosphate

CH2OPO3H,

I

CHOH

1
■■' CHOH

I
'■' CHOH

I
CO

I

CH20P03H2

Fructose-l, 6-PP

2. (x-carhoxylation

This takes place by a reversal of the oxidative decarboxylation catalysed
by an a-ketodecarboxylase. a-Carboxylation (giving pyruvic acid or
a-ketoglutaric acid, for example), contrary to jS-carboxylation, requires the
intervention of ATP, the general reaction being

R— C00-+ CO2 + 2H++ 2e + ATP ^

R— CO— COOH + P + ADP

The reversibility of the oxidative decarboxylation catalysed by an
a-ketodecarboxylase can be considered as possible, but not as established.

[b) Transmethylation

This is the term describing the transfer of a — CH3 group (a labile methyl
group) from one molecule to another. In order to distinguish methyl
groups attached by transmethylation from methyl groups originating in
other ways, compounds containing such groups which can be added or
removed in the presence of transmethylases are described as possessing
"labile methyl groups". Such compounds are choline, methionine, betaine,
sarcosine, adrenaline, anserine, methyl-nicotinamide, creatine, dimethyl-
glycine, etc. A compound containing a labile methyl group is not necessarily
capable of giving it up to an acceptor molecule. But, those compounds
which can do this are given the name methyl donors. Examples are choline,
betaine and methionine.

It would be wrong to believe that methyl donors, after activation by

BIOSYNTHESIS

233

CH3

CH3— NCH2CH2OH

/

CHa Choline

CH,

CHs— NCH2COO-
/

CHa Glycine-betaine or trimethylglycine

CH2SCH3

I

CHa

I
CHNH2

COOH

Methionine

S-adenosylmcchioninc

S-adenosylhomocysteinc

'%-CONH2

nicotinamide

-CONH2

methylnicotinamide

N

I

CHa

HNCH2COOH
I

C = NH guanidoacetic acid

I
NHa

CHa— NCH2COOH

I
C = NH

I
NH2

creatine

CH3>.

^N— CH2-CH2OH
CH3 dimethylethanolaraine

CHj

^N-CHi-CHsOH

CHa A^Tj choline

t^ria

Fig. 58 — Examples of transmethylation.

234

UNITY AND DIVERSITY IN BIOCHEMISTRY

transmethylases, form a common pool of methyl groups. On the contrary
each donor lies on a definite metabolic pathway :

choline -> betaine -> methionine ->■ acceptor

demethylation

gTycine-betainc

methionine <-f

I

choline s-adenosyimethionine

t

dimethylglycine

Precursors of Cj

\ COi

serine

— —— — ——— ^^ — — — — — — ->^ purin;

etc...

compourids containing
"^ lobile methyls

excretion

> = transmethylation
s z=^ =: activation
> = other reactions

Fig. 59 (Verly) — The methyl-group cycle. At each cycle, two out of three methyl groups
are lost, since the dimethylglycine formed by demethylation of betaine is not a methyl

donor.

In the last analysis, the labile methyl group always passes to the acceptor
from methionine. This is an operation requiring ATP. In fact, the true
donor is methionine activated by ATP, or S-adenosyl-methionine.

N-

:NHa

NHs C-N

\
CH

/ +

N=C— N— CH— CHOH— CHOH-CH— CHj— S— CH2— CHz— CH— COOH

I o I I I

CH3 NH2

S-adenosylmethionine

o

These labile methyl groups cannot originate from COg although their
oxidation can yield this substance. The precursors of labile methyl groups
are "formates" (Ci) and as in other cases where Ci fragments are required
the presence of folic acid and cyanocobalamine is necessary.

BIOSYNTHESIS 235

(c) Passage from Free Acetate to Active Acetate

We have seen how, in the priming reactions, active acetate or acetyl-CoA
is produced starting from various types of nutrient. Acetyl-CoA can also be
produced by activation of free acetate and this operation presents certain
curious features. In yeast and animal tissues the acetate is activated by
ATP and a special type of splitting of the molecule occurs, resulting in the
formation of AMP and pyrophosphate.

ATP 4- CoA— SH -^ CoA— S-PP + AMP

CH3— COOH + CoA— S^PP ^ CoA— S-'COCHs + PP

ATP + CoA— SH 4- CH3COOH ^ CoA— S-COCH3 + AMP -f PP

{d) Condensation of C^ Fragments to Form Acetoacetate

This is a modified Claisen condensation between two molecules of
acetyl-CoA resulting in the appearance of acetoacetyl-CoA and separation
of CoA. The reaction is reversible and the enzyme catalysing the conden-
sation also catalyses the reverse thiolysis. If a specific deacylase is present,
the acetoacetate is liberated and the equilibrium is distributed until the
acetyl-CoA is completely transformed into acetoacetate.

o o

II II

CH3— C— S— CoA + CH3— C— S— CoA
condensation \ \ thiolysis

OH O

CHs— C— CH2— C— S— CoA

S— CoA

o jr o

II II

CH3— C— CH2— C— S— CoA + HS— CoA

hydrolysis | -\- HoO

CH3— CO— CH2— COOH -f HS— CoA

{e) Biosynthesis of Isoprene and Carotenoids

Their biosynthesis is from Cg fragments by the intermediary of aceto-
acetyl-CoA. In isoprene, two carbon atoms are derived from the carboxy-
of acetate and three from its methyl, as shown below.

The carotenoids synthesized by plants also derive from acetyl-CoA.

236

UNITY AND DIVERSITY IN BIOCHEMISTRY

2 CH3— COOH -> CH3— CO— CH2— COOH

• o«o •©• o

CH3— CO— CH2— COOH -> CH3— CO— CH3 + COj

H.C

H.C

sO

C=0 + CH3

-COOH

H,C

H3C

C^

CH-

-COOH

jS-methylcrotonic acid

H,C

\o e o

-> C— CH = CH2

/
H2C Isoprene

Fig. 60 (Fukushima and Rosenfeld) — The synthesis of isoprene
O = carboxyl carbon • = methyl carbon

(/) Biosynthesis of the Steroid Ring System

The use of isotopes and mutant strains has demonstrated that acetate is
the essential starting material for the synthesis of the principal plant sterol,
ergosterol. Studies of cholesterol synthesis in animals have also provided
evidence of the importance of acetate. Neither formate nor labile methyl
groups (of methionine for example) can take part in the reaction, as
has been shown by the use of labelled carbon. It is most unlikely that
cholesterol obtains any of its carbons from a source other than the acetate
molecule. As shown in Fig. 61, by means of a series of chemical degrada-
tions it has been possible to demonstrate the origin of most of the atoms in
the skeleton either in the methyl or in the carboxyl of acetic acid.

HO

Fig

61 (Fukushima and Rosenfeld) — Origin of the carbon atoms of cholesterol.
# = derived from the methyl of acetate.
O = derived from the carboxyl group.

The bile acids, the sex hormones, the hormones of the adrenal cortex,
in animals, can all be obtained by modifying cholesterol and also directly from

BIOSYNTHESIS 237

acetate. In the case of the corticosteroids, experiment has shown that they
are formed from cholesterol molecules brought to the gland in the plasma.
The formation of cholesterol from acetate takes place through the inter-
mediate stage of acetoacetate, as has been shown in numerous experiments.

An examination of the distribution in the cholesterol molecule of the
carbons from methyl and those from the carboxyl of acetate, does not
immediately suggest the mode of synthesis. But it does limit the
possibilities.

The addition of a further acetic acid residue to the keto-group of aceto-
acetic acid, followed by a decarboxylation, yields a Cs branched chain
(three carbons arising from methyl groups and two from the carboxyl of
acetate) .

o

-.. COOH

'4-

o •
CH,C

+

o CH, .
OC ^
^CH,

'

'

•

o • o

^^

c-c— c

^c

It can be seen that this hypothetical C5 compound can be superimposed,
with respect to the origin of the carbon atoms, on the terminal part of the
side chain of the cholesterol molecule (Fig. 61). If we now consider the
distribution in the ring, we see that the latter can also be conceived of as a
polymer of the same C5 chain. It has however been objected that this makes
isovaleric acid appear to be a possible precursor of cholesterol. In reality,
this happens only by way of acetoacetate. Isovaleric acid (and also leucine)
gives an isopropyl residue which combines with CO2 to form acetoacetate.

What is the nature of the substance with the C5 chain? Let us recall the
synthesis of isoprene, the starting point for carotenoids and terpenes, from
acetate; we see that this synthesis leads to the C5 arrangement we are
seeking. Arguments have been advanced previously to implicate the iso-
prenoid, squalene, in the biosynthesis of steroids. Squalene is not limited
solely to the tissues of the selachians. Traces have been found everywhere
in animal tissues where it has been sought. When we consider that isoprene
units, as we have already indicated, are derived from acetate, we may
represent squalene as in Fig. 62 and compare it with Fig. 61.

238

UNITY AND DIVERSITY IN BIOCHEMISTRY

Fig. 62 (Bloch) — Likely distribution of carbon isotopes in squalene.

This hypothesis has been tested using labelled squalene and the results
have been favourable. However the ultimate mechanism of sterol formation
from squalene still remains undiscovered. The scheme shown in Fig. 63
has been proposed but part of this is still conjecture.

(g) Biosynthesis of Porphyrins

These substances are synthesized from 8-amino-levulinic acid formed
in the Shemin cycle (p. 222). Two molecules of the acid are condensed to
porphobilinogen, which is the precursor ring of the porphyrins.

COOH
I

COOH

CH,

1

CH,

1

CH,

CH,

c=o

• /

CH,

c-o

• /

CH,

1

NH,

NH,

-2H20

COOH

I

CH,

NHa

# = originally C^ of glycine.

Several suggestions have been made as to the mode of conversion of the
precursor to the different porphyrins. In order to account for the distri-
bution of the a-carbon of glycine and the S-carbon of amino-levulinic acid
in the porphyrins of series I and II, Shemin has proposed the following :
the condensation of three molecules of porphobilinogen forms a tri-
pyrrylmethane which is then split into a dipyrrylmethane and a mono-
pyrrole. The structure of the dipyrrylmethane will differ depending on
the place where scission has occurred. Fission at A or at B will produce the
types of dipyrrylmethane indicated by the letters A and B (Fig. 64).

J

BIOSYNTHESIS

239

The condensation of 2 molecules of dipyrrylmethane A will give a
porphyrin I and that of a molecule of A and a molecule of B will give a
porphyrin of series III (in this case with the loss of one carbon in the form
of formaldehyde) (Fig. 64).

d + Cz — CH3COCH2COOH + C2

-CO2

V

c=c-c

XJ

HO

HO

(fy^(Sj

Fig. 63 (Bloch) — Proposed scheme for the synthesis of cholesterol.

Ac Pr

/\

Ac Pr

CH,

I
NH,

H

NHjCH, H

Pr
Ac

H
■C-

Ac Pr

13

NH

CHjNH,

A '

Ac Pr

Ac Pr

CH. fi
NH|

JLc-O

H

CH,

I
NHi

H

"h,

'CH.NH,

Ac Pr

Pr Ac

Ac = chain of acetic acid
Pr = chain of propionic acid

Fig. 64 (Shemin) — • = Ca of glycine and Cs of amino-Ievulinic acid.

240

UNITY AND DIVERSITY IN BIOCHEMISTRY

(h) Biosynthesis of the Purine Ring

The use of radioactive isotopes has allowed the various carbon and nitro-
gen atoms in the purine ring to be identified with regard to their origin.
The purine ring cannot be formed in the living organism from
pyrimidines and as the diagram shows all the contributing fragments are
small units.

The biosynthesis of the purine ring takes place in the course of the
synthesis of the purine nucleotides, (p. 255).

C N

/ \ /'\
HaN C 5 3 CH

II

/ \3

HaN NH

4-aminoimidazole- 5 -carboxamide

COa

\

NH3 s

V. J'

y'.N

N/

C2

he

4C

r^ \^y-\^

NH,

Glycine

(i) Biosynthesis of Fatty Acids
The mechanism has been described together with the "fatty acid cycle"
(p. 196)-

BIOSYNTHESIS

241

(j) Biosynthesis of Sugars

The major process for the synthesis of sugars in Nature is the auto-
trophic synthesis which we shall be studying later (Part Six).

However, the synthesis of sugar molecules can, in general, be brought
about in the cell by a reversal of the priming reactions. In this way, in
certain types of cell, a fatty acid may give acetyl-CoA which does not enter
the tricarboxylic acid cycle but instead follows the reversed glycolysis
chain whose non-reversible stages are replaced by synthetic reactions the
enzymes for which are shown in Fig. 40.

Amino acids also can give molecules of hexoses and this "glucogenic"
property is particularly evident with those amino acids which enter the
cycle of priming reactions at the level of a-ketoglutaric acid and pyruvic
acid. Amino acids which enter at the level of acetoacetate can also, generally,
follow the same path, but they tend to bring about an accumulation of
acetoacetic acid and other "ketone bodies". They are therefore termed
"ketogenic".

As for the biosynthesis of the pentoses, we have already shown that this
can take place during the operation of the hexosemonophosphate shunt
(p. 193) and we shall return to other aspects of this biosynthesis when we
consider the formation of pentose phosphates (p. 253).

[k) Biosynthesis of Pyrimidines

The origin of the carbon and nitrogen atoms contributing to the structure
of the pyrimidine ring is shown in Fig. 65. The biosynthesis of the pyri-
midine ring takes place in the course of pyrimidine nucleotide biosynthesis
(p. 255).

NH,

C

6'

-^Nl \ 3C
\

CO.

aspartate

->-C2

4C

N

Fig. 65 — Origin of the carbon and nitrogen atoms in the pyrimidine ring.

242 UNITY AND DIVERSITY IN BIOCHEMISTRY

(/) Biosynthesis of Short Chain Amino Acids

Alanine is formed by transamination of pyruvate, and this latter sub-
stance is the starting material for the synthesis of other short chain amino
acids : glycine, serine, cysteine and cystine. According to Shemin, serine
is converted into glycine by way of formylglycine.

CHoOH

CHO

— 2H

+ H20

NHo— CH— COOH -^ NHo— CH— COOH

Serine

HCOOH + NH2CH2COOK

Formylglycine Glycine

The serine is synthesized from "formate" and glycine. If the formate is
tagged with C^* and the carboxyl of the glycine with C^^, doubly-labelled
serine is obtained :

HC^OOH + NHoCH^C^'OOH ^ NH^— CH— C^'OOH

The formation of serine from glycine and "formate" implies the partici-
pation of folic acid and pyridoxal, and this has been demonstrated by
several experiments with bacteria. A number of observations has shown
that the serine is derived from the glycolysis pathway and finally yields
glycine. The glycine can be formed not only from serine but also from
threonine with formation of acetate.

The carbon skeleton of cystine is also derived from serine, both in
mammals and in micro-organisms, as has been demonstrated by experi-
ments carried out with isotopes. Cysteine is actually an intermediate. The
sulphur of cysteine and cystine is derived from sulphates in micro-
organisms and from methionine in mammals.

Cysteine is readily transformed into cystine in the cell, according to the
equation

r^

OOH

2H

COOH

COOH

2 HCNH,

HCNH2

CHoSH + 2 H

Cysteine

HCNH2

CH2 — S — S — CH2

Cystine

BIOSYNTHESIS 243

The oxidation of cysteine to cystine is catalysed by cytochrome-c and
cytochrome-oxidase, whilst the reduction of cystine is accomplished by a
number of reducing agents : HgS, glutathione, SH-enzymes, etc.

The conversion of serine into cysteine is brought about by conden-
sation of the serine with homocysteine, formed by the demethylation of
methionine, or more exactly of S-adenosylmethionine, in the course of
transmethylation reactions.

CH3— S— CH,-CH,— CHNH,— COOH — HS CHj- CH^-CHNH.-COOH

methionine homocysteine

N CHj - CH, - CHNHi— COOH

"^ CH2 — CHNH2 - COOH HOCHj — CH NH, — COOH

cystathionine serine

HS CH2 - CHNHj - COOH HOCHj — CH,— CH NH, - COOH

cysteine homosenne

(m) Biosynthesis of Glutamic Acid

The chief source of glutamic acid is a-ketoglutaric acid produced in the
Krebs cycle. This acid can itself only arise in the oxidative decarboxylation
of isocitric acid, for the decarbox}dation of a-ketoglutarate to give succinate
is irreversible. This is the sole pathway for the formation of glutamic acid
from carbohydrate.

In certain micro-organisms like Escherichia colt, it is the only source,
whilst in other organisms other substances can contribute to the formation
of glutamic acid. These substances are proline, arginine, histidine, glycine
and succinic acid, by way of S -aminolevulinic acid.

The amino group is derived from ammonia fixed to a-ketoglutaric acid
by glutamic dehydrogenase, or from the amino group of another amino
acid, transferred by transamination.

[n) From Ghitamate to Glutamtne, Proline^ Hydroxyproline

and Arginine
Here, we have a biosynthetic pathway which, starting from a-keto-
glutaric acid in the Krebs cycle, divides at the level of glutamic acid
into three branches, one leading to glutamine, a second to proline and
hydroxyproline, and the third to ornithine, citruUine and arginine.

1. Ghitamine

Starting from glutamic acid and ammonia, in the presence of ATP,
glutamine is formed in a reaction whose mechanism is unknown.

244 UNITY AND DIVERSITY IN B lOCHEMISTK Y

2. Proline y hydroxyproline

The biosynthesis of proHne has been elucidated with the aid of strains
of E. coli which are specifically auxotrophic for proline. These strains fall
into two groups: those which accumulate glutamic y-semialdehyde and
those in which this substance can replace glutamic acid as a growth factor.
The reduction of glutamic acid to the semialdehyde is no doubt not so
simple as is indicated by the arrow in the scheme set out below. The
cyclization of proline would not require enzymic aid since y and S-amino-
aldehydes cyclize rapidly in neutral aqueous solution.

NH,
^"' ^ NHjCOCHjCHjCHCOOH

ATP glutamine

CH2 — Coj CH2 — CHa

I .11.

CHO CHCOOH ^ CH CHCOOH

/ \/

NHa N

glutamic y-semialdehyde J'-pyrroline— S-carboxylic acid

\

Cri2 — CHj

I I

CH2 CHCOOH

\/

NH

proline

Hydroxyproline is notably present in the connective tissue of animals.
In the latter, the use of isotopes has enabled it to be demonstrated that it
results from a modification of proline, which apparently is already present
since labelled hydroxyproline is not readily incorporated into the tissues.

3. Ornithine, citrulline, arginine

The formation of arginine from ornithine, via citrulline, has long been
known. In fact, the "ornithine cycle" of Krebs and Henseleit was one of the
first biosynthetic pathways ever proposed.

The sequence ornithine-citrulline-arginine, first shown to occur in
mammalian liver, has been found in many other organisms [Neurospora,
Penicillium, E. coli, lactobacilli, animal tissues, etc.).

The glutamate-ornithine relationship has been confirmed by the use of
isotopes and by the discovery of a Penicillium mutant responding to either
substance.

BIOSYNTHESIS

245

The use of mutants of E. coli has enabled Vogel to identify the three
acetylated compounds shown in the scheme.

Study of the pathway from ornithine to citrulUne, using mutants of
Neurospora, has shown that there are two stages. The first requires ATP;
CO2 and NH3 are fixed to form an as yet unidentified compound, which is
then transformed into citruUine.

GOGH

I

CH,
H.—

I

COOH

CH2

I
CHj —

CHO

CH,

I
CH, -

CHNHs

COOH

glutamic acid

CH,NH,

I

CH,

I
CH,

CHNH,

COOH

"cat

I

L

ornithine

CHNHCOCHj

COOH

N-acet}'lglutamic acid

NH,
\

CHNHCOCH,

COOH

y-semialdehyde of
N-acetylglutamic
acid

CH.NH,

I
CH,

I
CH, V

CHNHCOCH,

COOH
N-2-acetyl ornithine

NH

COOH

NH,

CNHCH

compound
X

t

I ATP
CO,

NHa

COOH

NH

I
CH,

c=o

/

NH

CH,

CH,

I
CH,

CHNH.

I

COOH

citrulline aspartic arginosuccinic
acid acid

CH,

I
COOH

NH

I
CH,

C=NH

COOH

I
+ CH

CHNH, CH,

I
CH,

COOH

1
CH,

CHNH,

I
COOH

CH,

I
CH,

CHNH,

I

COOH

arginine

CH

I
COOH

fumaric
acid

Fig. 66 (Davis) — Biosynthesis of arginine from glutamic acid.

The path from citrulHne to arginine is made up of several stages.
Citrulline is condensed with aspartate to form argino -succinate. A specific
enzyme cleaves the argino-succinate to arginine and fumaric acid.

(0) Biosynthesis 0/ C4 Amino Acids

[Aspartic Acid, Methionine and Threonine)

These molecules are derived from aspartic acid, itself formed from
members of the Krebs cycle. In plants and micro-organisms fumaric acid
is combined with ammonia in the presence of aspartase, whilst in mammals
which do not possess aspartase, aspartic acid is formed by reductive de-
amination of oxaloacetate in a reaction of unknown mechanism.

Chemical genetic experiments on Neurospora have shown that threonine
and methionine have a common precursor. In fact, a mutant requiring
both amino acids at once can satisfy this double requirement when
L-homoserine is supplied. On the other hand a mutant whose only block is
in the synthesis of methionine accumulates threonine and L-homoserine in
ts mycelium. The idea of a single precursor has been confirmed with

246

UNITY AND DIVERSITY IN BIOCHEMISTRY

E. colt and with yeast in isotopic experiments, and in enzymic experiments.
The synthesis of methionine is the reverse of the synthesis of cysteine
described previously (p. 243), via homocysteine and with cystathionine as

coon
I

CH.

I
CHNH.

I

coon

aspartic acid

CONH2
I
CHa

I

CHNH2
I
COOH

a>paragine

COOPO3H2 CHO

I I

CH2 TPN CH,

I I

CHNHj CHNH2

COOH COOH

j5-aspartylphosphate aspartic ^-semialdehyde

HO
I
CH3CH ■

NH,

CH-COOrt

CH20H

I

TPN or DPN CHj

CHNHj
I
COOH

homoserioe

ChreoDinc

(?)

NHj
I
CH3-CH2-CH-COOH

a — arainobutyric acid
CHaSH

CHNHj

COOH

cysteine

CHjSCH,

I
CHi

I

CHNH,

I
COOH

methionine

C,

CH2SH

I
CHj

I
CHNHj,

I
COOH

CH — S — CHi

CH2

I
CHNH2

I
COOH

CHNH2
I
COOH

homocysteine cystathionine

Fig. 67 (Davis) — Biosynthesis of C4 amino acids.

an intermediate (Fig. 67). (In micro-organisms cystathionine can be split
on either side of the sulphur atom. By contrast, in mammals it can only be
cleaved between the sulphur atom and the seryl residue. Hence mammals
cannot synthesize homocysteine.)

(p) Biosynthesis of Isoleucine, Valine and Leucine
The starting point of our knowledge of the mechanism of the biosyn-
thesis of these substances is the discovery of a Neurospora mutant requiring
both isoleucine and valine. This mutant accumulates the corresponding
dihydroxy-derivatives — dihydroxy-methylvaleric and dihydroxy-isovaleric
acids. Furthermore, a mutant of E. coli accumulates the corresponding
ketonic acids, a-ketomethylvaleric and a-ketoisovaleric acids. The mutants
accumulating the ketonic acids respond only to isoleucine and valine whilst
the accumulators of dihydroxy-acids respond not only to the amino acids
but also the the corresponding keto-acids. In an auxotrophe for isoleucine
alone, dihydroxy-methylvaleric acid and dihydroxy-isovaleric acid are
active, whilst in an auxotrophe for valine alone, dihydroxy-isovaleric acid
and a-ketoisovaleric acid are active.

BIOSYNTHESIS

247

methionine

CHjOH

I
CH,

CHNHi

COOH

homoserine

CH3

I
HOCH

^ CHNHa

alanine

a,/S-dihydroxy-
isovaleric acid

COOH

L-threonine

pyruvic
acid

CH3 CH3

\ /
COH

I
CHOH

I
COOH

CH3 CH3

\ /
CH

-CO,

CO

CH3 CH3

\ /
CH

CHa^-

CO

I COOH

COOH

a-ketoisocaproic a-ketoisovaleric

+ C2

acid

CH3 CH3

\ /
CH

I
CH2

i
CHNH2

acid

\

CH3

I
CHa

I
CO

transaminase

CH3 CH3

\ /
CH

CHNH2

COOH
a-ketobutyric
acid

CH,

I
CH,

I
:^ CHNHa

I
COOH

a-aminobutyric

acid

+

CH3

■ I
CO pyruvic

I acid

COOH

CH, CHo-COOH

\

COH

1 a,^-dihydroxy-^-methyl-

CHOH glutaric acid

I
COOH

CH3 CH2 — CH3

\ /
CH

I
CO

COOH
a-keto-j3-methylvaleric acid

CH3 CHj — CH3

\ /
CH

CHNH2

COOH

leucine

COOH COOH

valine isoleucine

Fig. 68 — Biosynthesis of leucine, isoleucine and valine.

248 UNITY AND DIVERSITY IN BIOCHEMISTRY

In addition, the synthesis of isoleucine and valine is blocked by the
absence of a single transaminase.

The sum total of facts indicates that a-ketoisovaleric acid yields leucine
by decarboxylation and condensation with a Cg fragment.

For the case of isoleucine and valine, the mode of biosynthesis is indi-
cated in Fig. 68,

(q) Biosynthesis of Amino Acids Derived from Benzene
{Tyrosine, Phenylalanine, Tryptophati)

The existence of mutants of E. coli and Aerobacter aerogenes requiring
for their growth the three amino acids containing the benzene ring, has
greatly aided the elucidation of the biosynthesis of these compounds. These
mutants require not only a mixture of the three benzenoid amino acids, but
also for the most part they require ^-aminobenzoate, ^-hydroxybenzoate
or a sixth factor, still unidentified. A large number of observations have been
made on mutants requiring the benzenoid amino acids — determination of
the substances accumulating in each case, study of competition between
compounds, etc. Further, at the enzyme level, comparative studies have
been carried out on vegetable tissues and micro-organisms, which syn-
thesize the benzene ring and on animal tissues which do not. The cofactors
of each enzyme, etc., have also been studied. All these investigations lead to
the conclusion that the intermediates in the synthesis of the benzene ring
are 5-dehydroquinic acid, 5-dehydroshikimic acid and shikimic acid. What
is the precursor of 5-dehydroquinic acid? On this point, so far, the mutants
have told us nothing. But a certain amount of information has been pro-
vided by the use of isotopes and from enzyme studies. This work shows that
the transformation of glucose into the benzene ring of tyrosine or phenyla-
lanine does not operate through the Krebs cycle. With the aid of labelled
glucose, using a mutant accumulating shikimic acid, it has been possible
to show that the carboxyl of this acid is derived from C-3 and C-4 of
glucose, the C-1 from C-2 and C-5 of glucose and C-2 of shikimic acid
from C-1 and C-6 of glucose. Hence the C-1 — C-2 — carboxyl portion
of shikimic acid comes from a degradation product of glucose, the four
other carbon atoms being of a more complex origin. Sedoheptulose-1,
7-diphosphate is an excellent precursor of shikimic acid and there are good
reasons to favour the theory which puts sedoheptulose as an intermediate
in the synthesis of the benzenoid amino acids.

The terminal stage of the synthesis has had some light cast upon it by
the results of enzyme studies. It has been shown that prephenic acid is an
intermediate in the path from phenylalanine to tyrosine. Moreover,
phenylpyruvic acid also lies along the pathway of phenylalanine
synthesis.

BIOSYNTHESIS

249

a

250

UNITY AND DIVERSITY IN BIOCHEMISTRY

When we come to tryptophan, which lies after shikimic acid, anthranilic
acid and indole are found to be intermediates in the synthesis. The con-
densation of indole with serine to give tryptophan is brought about by the
action of a specific enzyme, indole-ligase or tryptophan desmolase, whose
coenzyme is pyridoxal phosphate (this enzyme is not to be confused with
tryptophanase which splits tryptophan into indole and pyruvic acid).

In addition to the pathway just outlined, tryptophan can result from the
transamination of indole-pyruvic acid, but it seems unlikely that this
reaction makes any important contribution to the biosynthesis, A trypto-
phan-kynurenine-anthranilate-indole-tryptophan cycle has also been
proposed i.e. the reverse of the catabolic pathway described in Fig. 58.
However this sequence of reactions is only traversed if an excess of trypto-
phan is present and its function appears to be purely degradative.

(r) Biosynthesis of Histidi?ie and the Imidazole Ring
CH— NH CH— NH

Pentose (?)

^ C

-N

/

CH

CH

-N

/

CHOH

I
CHOH

I
CHaOPCH,

CH,

C=0

CH2OPO3H2

Imidazole-glycerolphosphate Imidazole-hydroxyacetonephosphate

CH— NH CH— NH CH— NH

CH

-N

/

^C

CH

-N

/

CH

-N

CH,

CH.

CH,

CHNH2

I
CHgOPO^H

CHNH,

30.12 CH2OH

Histidinolphosphate Histidinol

CHNHo

COOH

Histidine

Fig. 70 (Davis) — Biosynthesis of histidine.

Mutants of E. coli which are auxotrophic for histidine accumulate
L-histidinol, the corresponding aminoalcohol. Mutants of Neurospora have
been discovered since, which accumulate not only histidinol but also

BIOSYNTHESIS

251

imidazole-glycerol, imidazole-hydroxy-acetone and their derivatives phos-
phorylated in the terminal position of the side chain. It is these phosphory-
lated derivatives and free histidinol that are intermediates in the synthesis
whose terminal operation, the conversion of histidinol to histidine, is
catalysed by a DPN-containing enzyme which has been isolated from
E. colt. When we come to the imidazole ring itself, experiments with
sotopes have shown that C-2 is derived from "formate".

(s) Biosynthesis of Lysine
COOH

COOH

CH,

CHXOOH

Aspartic
acid

+

a — ketoadipic
acid

COOH

I
CHNH,

CH2

CH2

I
CH2

*CHNH2

*COOH

a — aminoadipic
acid

(moulds)

CH2NH2

CH,

CHg (bacteria)
CH,

CH,

CH,

CH,

CHNH2
COOH

lysine

CHNH2

COOH

a,a'-diaminopimelic
acid

Fig. 71 (Davis) — Biosynthesis of lysine.

The biosynthesis of lysine forms an exception to the general run of
biosynthetic processes we have so far considered and which we have been
able to assume, at least in general outline, to be the same in all cells. Here
the comparative biochemist has found a field of study at the level of a
biosynthetic mechanism. The biosynthesis of lysine, in fact, differs in
moulds and in bacteria. In the former, the starting materials are acetate and

252

UNITY AND DIVERSITY IN BIOCHEMISTRY

succinyl-CoA formed during the Krebs cycle. The intermediate stages are
a-ketoadipic acid and a-aminoadipic acid (which have been found in the
degradative pathway of lysine in the rat). In bacteria, one of the starting
materials is aspartic acid and a,a'-diaminopimelic acid is an intermediate.

B. Biosynthesis of Compounds Containing Ester,
OsiDE OR Peptide Bonds

(a) Biosynthesis of Complex Lipids

This subject has been extensively studied in the case of the lecithins
(phosphatidylcholines) and it has been shown that, in the presence of
certain enzymes of different specificity, the same mechanism is responsible
for the synthesis of cephalins (phosphatidylethanolamines). As with the
formation of glycerides, the starting point is a phosphatidic acid. The

HoCOH

(CH3)3

choline'
phosphokinase

NCHa — CH2OH
choline

ATP

(CH3)3 — NCH2 — CH20PO(OH)2

phosphorylcholine

cytidinetri-

phosphate

CTP

cytidinediphosphatecholine

HCOH

L- a-glycerophosphoric
acid

H2COPO(OH)2

+ 2RC0A-?

H2COR

I
HCOR

I
HaCOPOCOH)^

diacylphosphatidic acid

Fatty Aci<j[
cycle

H2COR

I
HCOR

i
H — C — H

1,2-diglyceride

L-a-lecithin

Fig. 72 — Biosynthesis of a lecithin.

knowledge that we possess today about the biosynthesis of complex lipids
stems from observations made on the incorporation of ^-P and ^^C-labelled
phosphorylcholine into the lecithin fraction of isolated liver mitochondria.
This incorporation is activated by CoA and ATP, and inhibited by metal
ions, in particular by Ca++. The incorporation requires a nucleotide as a

BIOSYNTHESIS 253

cofactor, cytidine-triphosphate. The enzyme is phosphorylcytidyl-
transferase and catalyses the combination of cytidine triphosphate with
phosphorylchoUne to form cytidine-diphosphate-chohne which is the
activated form of phosphorylchoKne. A molecule of cytidine monophos-
phate is released in this reaction, and in the presence of ATP is reconverted
to cytidine triphosphate. The phosphorylcholine itself is formed by a
direct phosphorylation of choline by ATP in the presence of the specific
enzyme choline-phosphokinase. But what is the acceptor of the activated
phosphorylcholine? The acceptor is a 1, 2-diglyceride, thus explaining
why natural lecithins are all of the a-type.

The 1, 2-diglyceride arises by a dephosphorylation of a diacyl-phospha-
tidic acid (a glycerophosphatide without the base) in the presence of a
specific phosphatase. The phosphatidic acids which are intermediates in
the biosynthesis of lecithins are the result of a combination, in the presence
of ATP and a specific enzyme, of a-glycerophosphoric acid with two
molecules of fatty acid activated by CoA. These compounds of aliphatic
acids and CoA are provided by the fatty acid cycle functioning in the direc-
tion of biosynthesis. The enzyme catalysing acyl transfer has a special
specificity for the acyl-CoA derivatives of the C^g and Cig acids.
Example:
palmityl-CoA + a-glycerophosphate

-> monopalmitylphosphatidic acid + CoA
palmityl-CoA monopalmitylphosphatic acid

-> dipalmitylphosphatidic acid + CoA

(b) Biosynthesis of Glycerides

It has long been recognized that the biosynthesis of glycerides occurs
via the intermediate formation of complex lipids.

The 1, 2-diglycerides acting as intermediates in lecithin biosynthesis
can also give triglycerides by the operation of an enzyme system present
in liver.

The following reaction has been observed in vitro in the presence of an
enzyme present in liver:

1, 2-diglyceride + palmityl-S-CoA -> triglyceride + Cox\-SH

This establishes an intimate relationship between the biosynthesis of
complex lipids and that of ternary lipids, in the first case, the reaction
being between a 1, 2-diglyceride and cytidinediphosphatecholine and in
the second case between the 1, 2-diglyceride and a coenzyme A. activated
fatty acid.

{c) Biosynthesis of Pentose Phosphates

As we have seen, D-ribose-5-phosphate is formed from G — 6 — P in the
hexosemonophosphate shunt.

254 UNITY AND DIVERSITY IN BIOCHEMISTRY

Another mode of formation is the condensation of C^ and Cg fragments to
form phosphoric esters of the pentoses and desoxypentoses, particularly
of desoxyribose.

This reaction

glyceraldehyde-3-P + acetaldehyde ^ desoxyribose-5-P

is a reversible aldol condensation, catalysed by desoxyribose-phosphate
aldolase. This enz3nne has been prepared from E. colt and numerous animal
tissues; the desoxyribose-5-P formed in the reaction is incorporated into
nucleosides.

As far as the metabolism of the pentose phosphates is concerned, the
principal reactions are those of transaldolization and transketolization.

A transketolase has been isolated from plant tissues and from animal
tissues, it catalyses the follow^ing reaction :

ribulose-5-P + ribose-5-P ^ sedoheptulose -7-P + glyceraldehyde-3-P

The enzyme is of low^ specificity and it also acts on ribulose-5-P, sedo-
heptulose-7-P, L-erythrulose, hydroxypyruvate and fructose-6-P. A rupture
of the ketol bond occurs and the "active glycolaldehyde" formed is
condensed w^ith an acceptor aldehyde. When the acceptor is glyceralde-
hyde 3-P, ribulose-5-P is formed.

(d) Biosynthesis of Oside Linkages

Although, theoretically, the enzymes catalysing the hydrolysis of oside
linkages should be capable of catalysing the reverse reaction of synthesis,
this is not considered to occur in practice.

In the case of sucrose, for example, the action of invertase causes the
reaction to go almost to completion from left to right. One of the substances
participating in the reaction, water, being present in overwhelming con-
centration, the hydrolysis of sucrose is in practice irreversible.

The synthesis of the osides operates, in fact, through their phosphory-
lated derivatives.

In plants, sucrose results from the condensation and the simultaneous
dephosphorylation of a molecule of phosphorylated glucose and a molecule
of phosphorylated fructose, in the presence of sucrose phosphorylase. A
system exists, and has been isolated from various bacteria, which, in the
presence of G — 1 — P and D-fructose, condenses these two molecules to
form sucrose vnth the elimination of phosphoric acid. This sucrose phos-
phorylase can combine glucose with, say, an aldose such as L-arabinose with
formation of a 1-3 oside linkage. Its specificity for the second half of the
molecule is hence seen to be of a low order. On the other hand, the sucrose
phosphorylase can utilize other sources than G— 1 — P as a donor of glucose
for the formation of sucrose. It is therefore also a transglucosidase capable of
transferring glucose derived from various donors, to a diversity of acceptors.

BIOSYNTHESIS

(e) Biosynthesis of Purine Nucleotides

255

The biosynthesis of these nucleotides, and consequently, of the purine
ring, begins with the formation of 5 '-phosphoribosyl-1 '-pyrophosphate
(phosphoribosylpyrophosphate, PRPP) which is the activated pentose
required in purine biosynthesis. Figure 73 shows the various ways in
which PRPP can be formed.

G-6-P

Pentose
cycle

oxidative
path

ribose

F-6-P

non-oxidative
path

ribose-5-P

ATP

ATP

kinases

kinase

-^PRPP

phosphomutase

ribose-1-P

nuc

phosphorolysis

eosides

Fig. 73 — Pathways of PRPP formation.

Starting with PRPP, in the presence of glutamine, 5'-phophoribosyl-
amine is formed. Addition of glycine and then of formate to the molecule
leads to formylglycinaminoribotide. The addition of CO 2 and the amino
group of aspartate gives the ribotide of aminoimidazolecarboxamide,
then an addition of a C^ fragment followed by closing of the ring gives
inosinic acid from which can be derived xanthosine-5 '-phosphate by
oxidation and guanosine-5 '-phosphate by oxidation and amination(Fig. 58).

(/) Biosynthesis of Pyrimidine Nucleotides

The carbon in the 1 -position and the nitrogen in the 2-position of the
pyrimidine ring are derived from NH3 and CO 2 which react in the presence
of ATP to form carbamyl phosphate.

256

UNITY AND DIVERSITY IN BIOCHEMISTRY

H2N

\^~

CH2— O — PO(OH)2
^C glycine

H H

H2C — NH2

I

^0 = C — NH — R— 5' — O — P0(0H)2

C — C

H I I H
OH OH

5 '-phosphoribosylamine

H2C

NH

\
CHO

glutamine

ATP

glycinamidoribotide

H2C — NH

\
CHO

HN = C— NH— R— 5'— O— P0(0H)2

formylglycinamidinoribotide

HC — N

0 = C— NH— R— 5'— O— PO(OH)2

formylglycinamidoribotide

\

aspartate, CO2 H2N — C = O

CH

ATP

•N

H2N — C — N — R — 5' — O — PO(OH)2
aminoimidazoleribotide

\
CH

H2N— C— N— R— 5'— O— PO(OH)2
aminoimidazolcarboxamide ribotide

xanthosine
monophosphate

+ H0O — 2H

HN — C = O

HC

C — N

adenosine
' monophosphate

aspartate

glutamine or NH3

\-

CH

guanosme
monophosphate

N — C — N — R — 5' — O — PO(OH)2

inosine monophosphate

Fig. 74.

BIOSYNTHESIS

257

O
H2N — C ~ O — PO(OH)2

carbamylphosphate

HN — C = O

I I
O = C CH

I II
HN — C — COOH

orotic acid

PRPP

^PP

HN — C = O

I I
O =C CH

1 II
N — C — COOH

R — 5' — O — PO(OH)2

orotidine — 5' — PO4

NHa

HN — CNH2

I I
O = C CH

1 II
N — C — COOH

I
R — 5' — O — OP(OH)8

aspartate

H3PO4

DPN

2H

3^

^

CO,

H2N COOH

I I

^ 0 = C CHa

I I

HN — CH — COOH

ureidosuccinic acid
(carbamylaspartic acid)

— H.O

HN — C = O

I I
O = C CH2

I I

HN — CH — COOH

dihydroorotic acid

HN — C = O

I I
^ O = C CH

I II
N — CH

R — 5' — O — PO(OH)3

uridine monophosphate

HN — CNH2

I I
->- O = C CH

I il
N — CH

I
R — 5 — O — PO(OH),

cytidine monophosphate

Fig. 75.

258 UNITY AND DIVERSITY IN BIOCHEMISTRY
ATP ^^ ^ COj + NHs

O

^ ^ ^^ II

ADP-f ^HaN — C - O — PO(OH)2

carbamyl phosphate

The carbamyl phosphate is condensed with a molecule of aspartate giving
ureidosuccinic acid, from which orotic acid is formed by cyclization and
oxidation. In the presence of PRPP and a pyrophosphorylase this acid
forms a ribotide and decarboxylation yields uridine monophosphate. The
decarboxylation of the product of amination of orotidine phosphate gives
cytidine monophosphate (Fig. 75). It can be seen that the pentose inter-
mediate in pyrimidine nucleotide biosynthesis is PRPP, the same as for
purine nucleotide biosynthesis.

(^) Biosynthesis of Nucleoside Diphosphates and
Triphosphates

This biosynthesis operates through the purine and pyrimidine mono-
nucleotides as follows:

nucleoside-P + nucleoside-PPP ^ nucleoside-PP + nucleoside-PP
2 nucleoside-PP ^ nucleoside-P + nucleoside-PPP
2 nucleoside-PP ^ nucleoside-P + nucleoside-PPP

(nucleoside and nucleoside designating nucleosides differing in the nature
of the base).

(/z) Biosynthesis of Dinucleotides

1. DPNandTPN

Nicotinamide can be formed by animal cells from tryptophan but is
usually present in the diet. Starting from ribose-1 -phosphate and niacin
(nicotinamide), DPN is probably synthesized as shown in Fig. 76. TPN
results from the phosphorylation of DPN in the presence of a specific
phosphokinase. TPN can be converted to DPN by a phosphatase.

2. FAD

Figure 77 depicts the formation of FMN, in the presence of a phos-
phokinase, from ATP and riboflavin, the latter a substance which is not
synthesized by the cells of the body and consequently must be obtained
from the diet. FMN is converted to FAD by stages analogous to those
governing the biosynthesis of DPN.

BIOSYNTHESIS

259

ribose-1-P

ATP

PP -*

nicotinamide

nicotinamide
riboside

nicotinamide
ribotide

*-DPN

ATP

ADP

ATP-

FiG. 76— Biosynthesis of DPN.

-riboflavin

ADP-<-

-»-FMN-

FAD-<-

Fig. 77 — Biosynthesis of FAD.

-ATP

-»-PP

pantothenic acid-

cysteine-

ATP

PP-^

pantothenylcysteine

CO,

pantetheine

-phosphopantetheine

ATP

>^ADP

— >- dephospho — CoA
(pantetheine — PO4 ~ PO4 — R — adenine)

ATP

CoA

ADP

Fig. 78 — Biosynthesis of coenzyme. A

260 UNITY AND DIVERSITY IN BIOCHEMISTRY

3. Coezyme A

Pantothenic acid is not synthesized by the body and therefore must be
obtained from the diet. It is condensed with a molecule of cysteine and
the resulting pseudopeptide is decarboxylated to form a compound of
pantothenic acid and thioethanolamine, pantetheine. The latter, in the
presence of ATP and a phosphokinase, becomes phosphopantatheine,
which, in the presence of ATP and a pyrophosphorylase, is converted to
dephospho-CoA, which, in turn, is phosphorylated on the 3' position of
ribose in the presence of ATP and a phosphokinase to form CoA (Fig. 78).

(/) Biosynthesis of Peptide and Amide Bonds

In animals hippuric acid is formed from benzoic acid and glycine which are
joined together by a secondary amide linkage similar to a peptide link. The
reaction has been well studied, ATP is required as an energy-donor and the
benzoic acid must be activated by being first combined with coenzyme A.

The stages of the synthesis are as follows: (E = enzyme)

(1) E + ATP ^ E— AMP + PP

(2) E— AMP + HS— CoA ^ E— S— CoA— AMP

(3) E— J— CoA + HOOC— CeHa ^ CoA— S— OC— CeHs +

E + H2O

(4) CoA— S—OC— CeHs + H2N— CH2— COOH^
CeHs— CONH— CH2— COOH + CoA— SH

(1+2+3+4)

CeHoCOOH + H2N— CHo— COOH + ATP ^

CeHs— CONH— CH2— COOH + AMP + PP

It is clear that during the synthesis ATP is split into AMP and PP.

A second type of synthesis of the secondary amide bond is found to occur
during the synthesis of pantothenic acid, a constituent of coenzyme A.
Here, we likewise have a splitting of ATP into AMP and PP, but not
through activation by CoA.

In the presence of an enzyme extract of E. coli we get
CH3

HO-CH,-C-CHOH-COOH + H2N-CH2-CH2-COOH + ATP

I
CH3

pantoic acid j3-alanine

CH3

-^HO— CH2— C— CHOH— CONH— CH2— CH0-COOH+ AMP + PP

I
, CH3

pantothenic acid

BIOSYNTHESIS 261

In the biosynthesis of glutamine from glutamate and ammonia it is the
y-carboxyl of glutamic acid which is activated, and the cleavage of ATP to
provide the 2500^000 calories for formation of the amide bond takes
place so that ADP and P are formed rather than AMP and PP. Glutamic
acid can also form amide bonds with hydroxylamine, hydrazine or
methvlamine.

The enzyme system responsible for the synthesis catalyses the following
series of reactions (E = enzyme) :

ATP

(1) E + ATP + Glu^E

\
Glu

ATP

/ Mg-H-

(2) E -f NH3 ^ Glu— NH2 + ADP + P -f E

\ Mn+ +

Glu

According to Bloch and co-workers, the enzymatic synthesis of glu-
tathione, requires only ATP

E + ATP - E— P + ADP

E— P + Glu ^ E— Glu + P

E — Glu -f Cys ^ E + Glu — Cys

E— P + Glu— Cys ^ E— Glu— Cys + P

E— Glu— Cys + Gly ^ Glu— Cys— Gly + E

In these four cases of formation of amide or peptide bonds, it is the
carboxyl group which is activated at the expense of one or the other of the
pyrophosphate linkages of ATP. The syntheses differ from each other in
the form of the activated carboxyl : bound to CoA, bound directly to the
enzyme or to the phosphorylated enzyme. But in no case has the activated
carboxyl itself been phosphorylated.

C. Biosynthesis of Macromolecules

{a) Polysaccharides

1. Synthesis of Glycogen

As already stated, glycogen is a branched polymer made up of D-glucose
units. Four enzymes are necessary for the degradation of glycogen to
glucose, and for the synthesis of glycogen from glucose. Two of these

262

UNITY AND DIVERSITY IN BIOCHEMISTRY

enzymes can act equally well in either direction, but the other two are
characteristic of the particular direction in which the reaction is taking
place ; this is shown in Fig. 79.

The first stage of the synthesis is phosphorylation by ATP, hexokinase
being the catalyst. It is here that a major expenditure of energy occurs; a
pyrophosphate bond (12,000 calories) forms a glucose-6-phosphate bond
(3000 cal) and by means of this reaction, which in practice is irreversible,
the glucose molecule is activated and prepared for a variety of metabolic
sequels. In the case we are considering, under the influence of phospho-
glucomutase an equilibrium is established between G — 6 — P and G — 1 — P,
in fact this equilibrium is part of the steady state in the cell and varies from
one cell to another as the steady state varies.

In the third stage, two enzymes act simultaneously, phosphorylase and
the branching enzyme. The glucose portion of a molecule of glucose- 1-
phosphate is added to the terminal glucose residue of a chain, with loss of
phosphate. The phosphate on Ci is exchanged for a glucoside linkage with
the fourth carbon atom of the terminal glucose residue, with little change in
free energy. In this synthesis the two substrates therefore are glycogen

glycogen + phosphate
phosphorylase I phosphorylase

amylo-(l ,4-*- 1 ,6)-transglucosidase
(branching enzyme)

amlyo-1 ,6-glucosidase

(unbranching enzyme)

G— 1— P (+ glucose)

ATP + glucose

oxidative
phosphorylation

phosphoglucomutase
hexokinase

phosphoglucomutase

G — 6 — phosphatase
G — 6 — P > glucose + phosphate

phosphohexose
-isomerase

ADP +P

\G — 6 — P dehydrogenase
\
\
\

6 — phosphogluconate

glycerol >• Fructose — 6 — P

lactic acid ?==^ pyruvic acid < amino acids

Fig. 79 (G. Cori) — Synthesis and degradation of glycogen in the liver.

and glucose -1- phosphate. It is the ratio of the concentrations of inorganic
phosphate and glucose- 1 -phosphate which determines the direction of the
reaction. Since, at pH 7*0, the equilibrium ratio for the reversible reaction
is 3 '2, the synthesis of glycogen will occur if there is more than one mole-
cule of glucose- 1 -phosphate for every three molecules of inorganic phos-
phate. Hence factors tending to lower the concentration of inorganic
phosphate will tend to favour synthesis taking place. When, in the branched
structure of the glycogen macromolecule, a side-chain has been formed

BIOSYNTHESIS 263

containing eight glucose units, then a new enzyme comes into play: this is
the branching enzyme. It is a transglucosidase, amylo-1, 4 -> 1,6-trans-
glucosidase, which converts a 1-4 linkage into a 1-6 linkage thus producing
a fork in the chain.

We see that whereas the concentration of glycogen in a cell can be
influenced by the requisite enzymes, by hormones and by other possible
regulators, the structure of the glycogen molecule depends on the relation
between the respective activities of phosphorylase, the branching enzyme
and the unbranching enzyme.

2. Synthesis of amylopectin

The so-called "Q-enzyme" is a transglucosidase which converts one
in every twenty 1-4 linkages of amy lose into 1-6 linkages to form a branched
chain. The enzyme only acts when at least forty-two glucose residues have
been united to form amylose. The formation reaction is the following :

a-D-glucose-1 -phosphate ^ amylose — ^ amylopectin

Like the branching enzyme, the "Q-enzyme" acts like a transglucosidase
capable of forming 1-6 bonds.

3. Synthesis of ^ linkages of polysaccharides

Fitting and Doudoroff (1952) have described an enzyme which in the
course of the synthesis of a glycoside linkage causes inversion of the type
of bond.

a-D-glucosyl-D-glucose -f P ^ ^-D-glucose-1-P + D-glucose

This demonstrates the possibility of the formation of complex poly-
saccharides containing /S-linkages by the action of enz^Tnes causing trans-
glucosidation.

(6) Proteins

By analogy with what is observed when amide bonds are synthesized in
peptides or amides, Borsook has suggested that during the synthesis of
proteins from free amino acids, it is the carboxyl of one of the amino acids
which is activated by utilization of a pyrophosphate linkage, the amino
group of the other amino acid not needing to be activated before formation
of the peptide bond. There are many arguments in favour of the idea that
proteins are synthesized from free amino acids. The activated amino acids
are transported on to a "template" where they arrange themselves in a
definite order. The peptide linkages are established for the price of the
energy of the pyrophosphate bonds and the molecule thus formed is
detached from the template.

264 UNITY AND DIVERSITY IN BIOCHEMISTRY

This mechanism impHes the activation, in the presence of ATP, of the
free carboxyl group of the amino acids which go to form the new protein
molecule. This idea is supported by a large number of experimental obser-
vations. The activation of the carboxyl group is brought about by specific
enzyme for a particular amino acid, which first attaches it to AMP. The re-
sulting amino acid-AMP-anhydride is then joined to a short soluble piece
of an RNA chain, the whole taking place on a complementary sequence of
an RNA template.

1. The ijitracellular pool of amino acids

The existence of an intracellular pool of amino acids not combined as
protein, either free or in the form of amides, etc., is now generally admitted.
In vertebrate tissues, the amounts of these non-protein anmino acids, acting
as a source for protein synthesis, are small. On the other hand they are
much greater in the tissues of such animals as the marine crustaceans, and
in plant cells, in yeast cells, or in the cells of certain Gram-positive bacteria.

The factors regulating the total amount and composition (which in
animals appears to be specific for each tissue of a particular species or, for
a given tissue, specific for each species) of the intracellular pool of amino
acids, are of special interest. The composition of the pool of non-protein
amino acids differs from the overall composition of the proteins in the cell
which contain it, and this has been demonstrated in widely different cases,
for example in animal cells and in infusoria.

A particularly detailed study of the pool of non-protein amino acids in
yeast was carried out in Spiegelmann's laboratory and has given some
highly interesting results. It is possible to modify the composition and the
amount of the intracellular pool by various changes in the culture medium.
If the medium contains no nitrogen and contains glucose, the intracellular
pool in the yeast decreases in amount and this decrease affects all the
constituents. If the cells are then placed in a medium containing both
glucose and nitrogen the pool is replenished, but in a manner which differs
according to the nature of the source of nitrogen. If the nitrogen is provided
by a casein hydrolysate the pool replenishes all its constituents. If ammon-
ium chloride is the source the restoration is much slower and certain amino
acids such as methionine, threonine, proline, lysine and histidine, only
return to their former concentration after some considerable time. On the
other hand, irradiation with ultraviolet light increases the amount of the
pool in the yeast cell, and a considerable number of observations seem to
indicate that in yeast cells there is an internal mechanism for replenishment
of the pool which depends upon the degradation of a labile protein
compound.

In Staphylococcus aureus, which has been studied from this aspect by
Gale, the synthesis of a large number of amino acids is not possible and

BIOSYNTHESIS 265

they must be obtained from outside the cell. If the medium is rich in amino
acids, they accumulate in the cell during the period of growth and the
intracellular pool is increased. However, if protein synthesis is rapid and
normal, the amino acids do not accumulate.

2. The template

A large number of experimental facts and a series of indirect experiments
have led Brachet and Caspersson, independently of each other, to the
conclusion that nucleic acids are involved in protein synthesis. The con-
centration of RNA in the cell is approximately proportional to the growth of
Bac. lactic aerogenes, thus leading Caldwell to consider RNA as being the
template itself. Jeener has shown for his part that during experimental
modifications of the volum.es of the nucleus and the cytoplasm of Thermo-
bacteriiim acidophilus protein synthesis was quantitatively related to the
level of RNA. Finally, direct experiments have shown that in various cells
or fragments of cells, treatment with ribonuclease suppresses protein
synthesis.

HbA Val— His— Leu— Thr— Pro— G/«— Glu— Lys

HbS Val— His— Leu— Thr— Pro— Ffl/— Glu— Lys

HbC Val— His— Leu— Thr— Pro— Lj'^— Glu— Lys

HbG Val— His— Leu— Thr— Pro— Glu— G/j— Lys

Fig. 80. (Perutz). Sequence of amino acid residues in a small segment of OHe of the

polypeptide chain of haemoglobin.

These are the facts which point to RNA being the template on which
the synthesis of proteins takes place. Another confirmation has been
brought by experiments showing that the isolated nucleic acid of tobacco
mosaic virus can introduce the disease into a leaf cell as well as the whole
nucleoprotein of the virus.

3. The synthetic process

It is now a well established fact that the system of activation of the dif-
ferent amino acids in the presence of the specific enz}'Tne for each of them,
is the same in all cases of protein synthesis, and that the characteristic
structure of the protein synthetized is due to the structure of the RNA
template. This template is located in the cytoplasm. It is probably syn-
thetized in the nucleus where the coded message for the synthesis of a
particular protein is transferred from a section (or gene) of a long DNA
macromolecule to a corresponding section of RNA. Each amino acid is
brought to the template by a specific carrier. Crick has suggested that this
carrier is a short length of RNA chain with a specific sequence of bases
coding for the particular amino acid. It has been shown that each specific

266

UNITY AND DIVERSITY IN BIOCHEMISTRY

enzyme catalyzing the activation of a particular amino acid in the presence
of ATP, with the formation of an amino acid-AMP-anhydride, also
catalyzes the fixation of this compound on a short, soluble piece of RNA
chain. This carrier brings the amino acid on to the complementary sequence
of the template. By this mechanism, the long code represented by numerous
genes on a DNA chain, copied on RNA chains, controls the biosynthesis
of a number of proteins, among which are numerous enzymes controlling
the specific metabolism of the cell.

porphyrins

serine (C — P)

CH3—

histidine (C— 7)

(methionine)

CO2

purines

Ci

CO.

purines

oxalosuccinic acid
oxaloacetic acid
arginine (guanidine C)
glucose (C — 3 and 4)

histidine (C — 1 and 5)

t
pentoses < v

oxaloacetate

-►-pyrimidines

methionine (C — 1 and 4)

desoxypentoses -^-

phosphoglyceraldehyde

aspartic->-homoserine

acid

isoleucine (C — 3 and 4)
valine (C— 3 and 4')

threonine

cysteine

\.

serine-^ — glycine-*'
cystine

pyruvic acid

/

a-aminobutyric
acid

isoleucine
(C— 1,2,5 and 6)

alanine
tr>-ptophan side
-chain valine (C — 1,2 and 4)

leucine (C — 3,4,5 and 5')

succinyl-CoA

a-ketoglutarate

steroids, leucine (C — 1 and 2)
carotenoids, lysine (C — 1 and 2)
terpenes, oxaloacetic acid, fatty acid

-►-porphyrins
lysine

-glutamic acid

lysine (C — 3 and 6)

proline

sedoheptulose

i

i.

hydroxyproline

benzene ring of
amino acids

Fig. 81 — Utilization of various key-materials for biosynthesis.

BIOSYNTHESIS

267

4. The transmission of the code

We have seen that the macromolecule of DNA is a double hehx in the
form of a spiral staircase in which the links between the different nucleic
acids form the banisters and the purine and pyrimidine bases form the
steps. As we have seen, each step is formed by one of the two combinations
adenine-thymine (or thymine-adenine) and guanine-cytosine (or cytosine-
guanine). This means that a particular sequence of bases on one of the
banisters is paired to one definite complementary' sequence on the other.

Now if the chains separate in a medium containing free nucleotides of
deoxyribose and one of the bases (adenine, guanine, thymine or cytosine),

formic acid
formaldehyde
methanol
glycine (a — C)
glycollic acid (a — C)
serine (a and /3 — C)

threonine (a — C)
histidine (C — 2)
tryptophan (C — 2)
phenylalanine (^ — C and C — 3)
tyrosine (j8 — C and C — 6)
acetone

Cx

glycolysis

/

phosphoglyceraldehyde

<

\

\

pyruvic acid

acetyl-CoA

fatty acids
pyruvic acid
amino acids

acetic acid
ethanol

r^\

\

oxaloacetate

'■ 1 \/

succmyl-CoA | f

/

a — ketoglutarate | /

tricarboxylic
acid cycle

sedoheptulose

t

hexose monophosphate shunt

Fig. 82. The chief sources of the key-materials required for biosyntheses.

each of the two chains of the parent double helix will become a template for
a complementary chain, the result being the formation of two daughter
double helices, carrying an exact copy of the code. How the sequence on

268 UNITY AND DIVERSITY IN BIOCHEMISTRY

bases in one of the chains, copied on a RNA chain, is read in terms of
amino acid succession, remains to be explained. Crick, Griffith and Orgel
have devised a code in which the names of the twenty amino acids are read
in terms of twenty triplets of bases, but this ingenious suggestion has not
yet been experimentally confirmed.

We have said that we know at present a number of mutants of micro-
organisms, characterized by the lack of a definite enzyme. Other mutants
are characterized by the addition of a new enzyme. In each case we are
dealing with changes in structure of protein molecules resulting in the
loss or in the acquisition of the enzymatic activity.

This leads to the notion that, in certain cases at least, a mutation is
primarily a change in the structure of a protein molecule, i.e. an aspect of
the molecular evolution (heteromorphic evolution, p. 336) of proteins.
Abnormal haemoglobins show that the alteration of a single residue may
deeply change the properties of a protein macromolecule. In this case, a
single mistake in copying the genetic code during its duplication may lead
to a permanent change in the properties of haemoglobin. Each half molecule
of haemoglobin is formed by about 300 amino acid residues. Figure 80
shows, in a definite small segment of this unit, the sequence of amino acid
residues in normal haemoglobin (HbA), in sickle cell haemoglobin (HbS),
in haemoglobin C disease (HbC) and in haemoglobin G disease (HbG).
Figure 80 makes it quite clear that, in each of these diseases, only one
residue, printed in italics, is altered.

D. BlOSYNTHETIC INTERRELATIONS

The same remarks which were made about the pathways of complete
degradation apply also to the pathways of biosynthesis.

The biosynthetic pathways described here are the longest paths it has
been possible to trace. These paths are not always traversed from end to
end and they can be entered at numerous junctions. The existence of
transferring enzymes and the reversibility of many sections of the metabolic
routes, both degradative and synthetic, establish multiple interrelations
between the routes for biosynthesis and those for degradations.

REFERENCES

Anfinsen, C. B. (1959) The Molecular Basis of Evolution, John Wiley and Sons,

New York.
Block, K. (1954). Biological synthesis of cholesterol. Harvey Lectures, 48, 68-88.
Davis, B. D. (1955). Intermediates in amino acid biosynthesis. Advanc. EnzymoL,

16, 247-312.
Greenberg, D. M. (1954). Chemical Pathways of Metabolism, 2 vol. Academic

Press, New York.

BIOSYNTHESIS 269

McElroy, W. D., and Bentley Glass, H. (Editors), (1955). A Symposium on
Ammo Acid Metabolism, Johns Hopkins Press, Baltimore.

OcHOA, S. J. B., (1952). Enzymatic mechanisms of carbon dioxide fixation. The
Enzymes, Sumner, J. B., and Myrback, K. (Editors), vol. II, part 2. Academic
Press, New York.

Perutz, M. F. (1959). The molecular basis of life. Research, 12, 326-334.

PART FOUR

TOPOBIOCHEMISTRY AND CELLULAR
REGULATION

CHAPTER I

CELLULAR TOPOCHEMISTRY

It would be a grave mistake to believe, as is often stated, that the cellular
theory of life, as proposed by Theodor Schwann, was of a purely morpho-
logical character, Schwann not only affirmed that "Everything which lives
is made up of cells", but he also pointed out that the individual life of each
cell "has its origin in forces which are inherent in each molecule". For his
teacher Johannes Muller, the phenomena of life were the result of an idea
acting on each tissue producing in it a "vital energy". On the other hand,
Theodor Schwann stated that living phenomena are the product of forces
which are essentially the same as those present in inorganic nature, forces
acting blindly and compulsorily like physical forces. The forces which
bring about the formation of organisms, Schwann added, do not act in
non-living nature because the combinations of molecules which give rise
to them do not occur there. But, he added, it does not follow that it is
necessary to distinguish them from ordinary physical or chemical forces.
In his work Mikroskopische Untersuchungen, Schwann proposed the classi-
fication of tissues which is still to be found, without major alterations, in
text books of histology. But he was forced to watch in despair the develop-
ment along purely morphological lines of the microscopic anatomy which
he had founded. The never-ending controversies among the histologists
and cytologists over their artifacts of coloration and fixation were of no
interest to him. To Schwann, the pressing problem was the study, by
physical and chemical methods, of metabolism (using the word he had
created) of units smaller than the cell itself. But, he had arrived too
soon, in a world too immature and only capable of producing great numbers
of publications illustrated by beautiful colour pictures of tissues. This
enormous mass of literature has left us little else than the idea of the
presence in the cell of a nucleus containing a nucleolus, and a cytoplasm
in which one can distinguish the presence of mitochondria and other in-
clusions. Classical histology, as the fruit of a hundred years of tissue-sHcing,
fixing and staining, has provided us with a great number of pictures of
cells, pleasantly coloured with Janus green or gentian violet. Unfortunately
they bear no relation to the complex phenomena described, in an abbre-
viated and simplified form, in the preceding pages. One might as well try

273

274 UNITY AND DIVERSITY IN BIOCHEMISTRY

to explain a watch to a Martian by drawing him a small circle, even though
it have the agreeable brightness of gold.

It was necessary to wait until 1940 when Albert Claude had the idea of
dissecting the cell into its constituents by means of a centrifugal field
applied to a suspension of macerated cells under defined conditions. This
mode of penetration into the unexplored world inside the cell gave access
to previously recognized organelles and led to the identification of others,
as was the case with the microsomes. The parallel use of the electron
microscope gave further valuable help in this field. Once more the march
of scientific progress continued. Schwann would not have formulated the
cellular theory if he had not had the benefit of a microscope incorporating
improvements introduced by Amici. Similarly, without the ultracentrifuge
and the electron microscope, Claude, and many workers after him, would
not have been able to penetrate inside the cell.

From a biochemical point of view, a cell can be divided into a nucleus and a
cytoplasm containing mitochondria and other inclusions. Even with the or-
dinary microscope, and better still with the phase-contrast microscope, it is
possible to distinguish granular or fibrillar inclusions in the cytoplasm. In
cells which respire, mitochondria are found, and in certain cells there are other
specialized structures buried in an apparently structureless cytoplasm.

A. Cytoplasm
(a) The Fundamental Material

The C)rtoplasm has been the subject of numerous theories. The most
recent have been thought out with a view to explaining the physical
properties of cytoplasm in terms of its structure. The most comprehensive
is the theory of bonds, proposed by Frey-Wyssling.

During the first quarter of this century, ideas about the structure of
protoplasm were dominated by a fallacious conception which sought to
interpret the properties of protoplasm according to whether the material
was in the sol or the gel state, where these terms have the same significance
as in colloidal chemistry. Today, a large number of facts make us consider
that the cellular elements are formed from macromolecules and not from
small molecules associated together by physical forces to form micelles
analogous to those found in soaps.

The cell is made up of a number of organelles bathed in a cellular juice.
The cytoplasm contains protein filaments which make up a labile frame-
work whose structure is due to bridges between the fibres and whose
lability is due to the fact that the bridges are easily broken. The filaments
themselves vary between 80 and 200 A in thickness.

If we consider the number of different amino acids present in proteins,
we can understand what a variety of side-chains is possible in a polypep-
tide chain.

CELLULAR TOPOCHEMISTRY 275

The complex macromolecule represented by the network of protein
fibres in the cytoplasm is maintained intact by a series of junctions between
polypeptide chains (see p. 99). Certain of these linkages are homopolar in
nature, for example the disulphide bonds. Others are joined by heteropolar
bonds, or salt linkages. In addition there are cohesive bonds between non-
polar groups (for example attraction between CHg groups) and polar groups
(for example attraction between groups of a dipole nature).

These various linkages are shown in Fig. 83.

According to the theory of Frey-Wyssling, the four types of bond occur-
ring between the peptide chains can explain the behaviour of cytoplasm.

1 . Cohesive bo?ids between non-polar groups {binding by Van der Waals forces)

These are the same forces as are responsible for the cohesion of a crystal
of a paraffin. The attraction between non-polar hydrocarbon groupings,
feeble as it is, cannot be accounted for by an electric field for such a field is
practically non-existent. (In substances containing lipophilic groups it is
difficult to explain the attraction of these groups since their electric field
of force is negligible when compared with that between polar molecules.)
The cohesion between methyl groups, for example, is very small and
temperature-sensitive. The change in viscosity of cytoplasm with tempera-
ture must be attributed to the presence of these bonds.

2. Cohesive bonds between polar groups

These are bonds due to residual or secondary valencies and are caused
by dipole interaction. They are semi-chemical in character and they are
commonly called hydrogen bonds (Pauling) (see p. 99). When a polar group
carr}dng a peripheral hydrogen atom ( — OH, — NHg, etc.) is present, it can
be attracted electrostatically by the negative charges located on the polar
groups of neighbouring molecules thus uniting them through a hydrogen
atom. If the polar groups of the two adjacent molecules cannot approach
each other sufficiently, the electric field between them will attract water
molecules. In this case the bridge is made up of attracted water molecules
and the linkage is sensitive to any swelling of the cell, an action depending
on the presence of inorganic ions. Small ions like Li+ or Na+ have a
hydration shell thicker than that surrounding larger ions like K"*" or Cs^.
As the size of an ion increases with its atomic weight the hydration shell
decreases correspondingly. Hence it can be seen that the introduction of
various ions can modify the hydration of such structures as cytoplasm.

3. Heteropolar valencies (salt linkages)

If the positive and negative ends of two side-chains are in proximity
they can enter into a salt linkage, their charges neutralizing each other and
the degree of hydration decreasing. To break such linkages it is necessary
to change the pH.

276

UNITY AND DIVERSITY IN BIOCHEMISTRY

Fig. 83 (Frey-Wyssling) — Types of link between neighbouring polypeptide chains.

O = a water molecule.

4. Homopolar valencies

These can be formed in a variety of ways : by elimination of water
(formation of ester, glucoside or peptide linkages), by removal of hydrogen
(formation of disulphide bridges, methylene bridge, etc.), they are in-
fluenced by the oxido-reduction potential of the system.

The theory of Frey-Wyssling postulates the existence of monomolecular
polypeptide filaments which must be about 20 A thick. However the
electron microscope has revealed the presence in the cytoplasm of an
endoplasmic reticulum made up of trabeculae 30 to 40 vayi in length, hollow
tubes joining together vesicles 100 to 300 nijx in diameter. Attached to this
system there are basophilic particles. In addition the cytoplasm contains
fibres either singly or in bundles, which make up the contractile material
of the cell and by means of a slow but continuous motion maintain the
various organelles suspended in the cellular fluid.

Molecules such as the phosphatides can react with the protein side-
chains regardless of whether they are lipophilic or hydrophilic in nature, as
shovm in Fig. 84.

<

^ O C 0 0 o

■ CH,
. CH,

CH,

V O 0 0 ° 0

>

CM,

• OH

O 0 c '

CH. <

Lecithin

CH, -

Glyceride

Lecithia

Polypeptide chain
Fig. 84 (Frey-Wyssling) — o = a water molecule.

CELLULAR TOPOCHEMISTRY 277

As for glycerides, which do not possess a hydrophilic group, they can only
attach themselves to lipophilic side-chains and then, as shown in Fig. 88,
only by the interposition of a phosphatide or similar molecule. The sterols
have a polar structure analogous to that of the phosphatides. But the phos-
phatides are more reactive for their hydrophilic end bears an ionized
phosphate group (negatively charged) and a positive ammonium group.
By contrast the sterols can only form esters.

From the point of view of enzymology, cytoplasm is a not highly organ-
ized multi-enzyme system containing notably all the enzymes of the
glycolysis system and which consequently is qualified for the formation of
ATP energy-rich bonds. In addition to the conversion of sugars into pyru-
vic acid, cytoplasm can saponify lipides and split proteins into amino acids.
In a cell living anaerobically, 80-90% of the glycolysis occurs in the
cytoplasm.

(b) The Outer Region of the Cell

In all cells it is possible to distinguish an inner region and an outer
region. In certain cases, such as the amoeba, the region of clear ectoplasm
is plainly differentiated from the granular endoplasm.

The ectoplasm is itself surrounded by a thin layer of polysaccharide and
lipide-containing material called the plasmalemma. This portion is elastic
and contractile. The difference between the endoplasm and ectoplasm is a
result of differences in the shapes of the constituent macromolecules.

We may represent the cellular "membrane", from the interior to the
exterior, as formed, firstly, of a highly hydrated protein structure consisting
of leaflets parallel to the surface, this explains its contractility and elasticity.
Beyond this membrane are situated one or two layers of complex lipides,
followed by a layer containing polysaccharides.

The presence of enzymes in the outer region of the cell has been demon-
strated, chiefly with the aid of indirect methods. Thus, a portion of sub-
strate can be introduced to see if the requisite enzyme is present ; this has
been done for certain esterases, phosphatases, etc.

(c) Microsomes

There is always present in cytoplasm an important basophilic material
which forms the endoplasmic reticulum of vesicules and canaliculi. At
least part of this material appears to consist of the particles which we call
microsomes. The discovery of microsomes (50-150 ma in diameter) is due
to Claude, who isolated them from a mush of cells by prolonged centri-
fugation at 20,000 rev/min. The microsomes contain a large part of the
nitrogen of the cell and a comparatively (more than the mitochondria) large
fraction of the phospholipides and RNA. It was at one time considered that
the microsomes consisted of mitochondrial fragments, but this view is no

278 UNITY AND DIVERSITY IN BIOCHEMISTRY

longer tenable for it has been shown that they are the exclusive carriers
of certain enzymes like glucose-6-phosphatase and they are without the
typical enzyme of the mitochondria, cytochrome-oxidase. As already stated,
it is believed that protein synthesis takes place under the influence of
ribonucleoproteins. The microsomes, amongst the other cellular organites,
are the most abundantly provided with these substances, and this is why
Brachet considers that the microsome, rich in RNA and incorporating
amino acids very rapidly, is the probable site of protein synthesis.

The enzymatic composition of microsomes is still obscure. Nevertheless
we know that they contain important amounts of cytochrome-reductases.
They must therefore presumably play some part somewhere in the stage
of electron transfer between DPNH or TPNH and cytochrome-c.

In addition, according to Brachet, they contain a number of hydrolases :
phosphatases, amylases, cathepsin, and ribonuclease. Several experiments
also indicate the presence in microsomes of the enzyme system responsible
for the incorporation of alanine into proteins.

B. Mitochondria

We may say, and rightly, that the mitochondria is the engine which drives
the cell. For although the energy-rich bonds of ATP are formed in the
cytoplasm during glycolysis, the major production results from oxidative
phosphorylations which take place in the mitochondria. We may also liken
them to the central bank for the cell for it is from here that the currency
in which the cell deals, is issued.

The mitochondria are cylindrical particles 1^ [jl in length and 0-3-0 -7 [x
thick. They are bounded by a double membrane of protein in the middle
of which there is a double layer of complex lipides. In mitochondria from
mammalian liver and kidney, each protein layer is 45 A thick and the
distance between the two protein layers is 70 A, so that the membrane
surrounding the mitochondria is 160 A thick. This membrane is prolonged
into the interior of the mitochondria by ridges penetrating into the sub-
stratum which appears to consist mainly of soluble proteins.

The general features of mitochondria have been found in all the cells
of the different and varied organisms in which they have been studied :
mammals, birds, batrachians, molluscs, annelids, protozoa, yeasts, plants, etc.

In mitochondria from rat liver the greater part (56%) of the organic
phosphorous is in the form of complex lipides and 20% is present in RNA,
the only nucleic acid they contain. A quarter of the total nitrogen of the
liver cell is in the mitochondria and about 15^o of the RNA of the cell is
found there.

Cytochrome oxidase or cytochrome a^, appears to occur only in the
mitochondria. However, this is not the case for the cytochrome-reductases
(DPN and TPN specific) which, as stated above, are also found in the

CELLULAR TOPOCHEMISTR Y 279

microsomes. Succinic dehydrogenase, fumarase, and the oxido-reduction
systems for a-ketoglutarate and oxaloacetate are also found only in the
mitochondria, which contain in addition the enzymes of the fatty acid cycle.

Their enzymatic equipment shows that the mitochondria are the ex-
clusive sites of aerobic oxidation and oxidative phosphorylation.

In addition to these heavy mitochondria, the exclusive carriers of
cytochrome oxidase, de Duve distinguishes "light mitochondria" or
"lysosomes", which contain alkaline phosphatase, ribonuclease, desoxy-
ribonuclease, "cathepsin" (i.e. the complete assembly of intracellular
peptidases), and most of the ^-glucuronidase. In these intact particles,
these hydrolases do not have access to their substrates when the latter
are situated in the medium around the particles.

C. The Nucleus

The cellular nucleus, during interphase, consists principally of a nuclear
sap or caryolymph, a nucleolus and certain chromatin filaments or chro-
monemata which condense into chromosomes during mitosis.

In the nucleus we find a whole series of compounds. The nuclear sap
contains proteins not belonging to the histone or protamine families. In
addition, we find in the nucleus either protamines or histones, depending
on the cell, and also lipides (around 10%).

The nucleus contains practically all the DNA in the cell. For a given
species, the amount of DNA per nucleus of diploid cells in interphase is
constant. Although DNA in each case is a complex mixture of different
nucleotides, its composition is the same for all the different cells of the
organism, for they are all derived from the same fertilized &gg. It differs
from one species to another. Two insect viruses parasitic on the same
organism, for example, will have different structures of their DNA. A cell of
E. colt and its bacteriophage have DNA of differing structures.

The fact that a spermatozoid contains only half the amount of DNA
present in normal cells clearly identifies DNA with the chromatin filaments
which also contain protamines and histones.

However, the nucleus also always contains some RNA located in the
nucleolus (which does not contain DNx\) and in the chromatin filaments.
The ratio RNA/DNA varies from one species to another and, in the same
species, from one organ to another. Unlike DNA whose composition is
characteristic of all the cells of a species, the composition of RNA varies
in the same cell from one point to another. Thus the RNA of the nucleus,
the microsomes, and the mitochondria are all of different composition.
Physiological conditions can also modify the composition of the various
types of RNA in an organism, although they have no influence on the
composition of the DNA.

280 UNITY AND DIVERSITY IN BIOCHEMISTRY

The nucleus contains a number of other phosphorylated derivatives and
inorganic elements such a K, Ca and Mg.

When mitosis occurs, the chromatin filaments combine into chromo-
somes which divide longitudinally and then distribute themselves between
the two halves of the cell. This process is aided by the aster, a structure
formed at the beginning of mitosis and surrounding the centrosome. It has
been isolated from sea urchin eggs and shown to be protein in nature.

The nucleus is lacking in a number of important enzymes for cellular
oxidation such as cytochrome oxidase, succinic dehydrogenase, uricase,
D-amino acid oxidase, etc., and so respiration does not occur. However, it
does contain a glycolytic system, although of low activity. In the nucleus
is concentrated a system of enzymes for the synthesis of nucleic acids and
nucleotides. It is found that radioactive phosphorus is always incorporated
more rapidly into the RNA of the nucleus than into the RNA of the cyto-
plasm. So, there is little doubt that the nucleus contains enzyme systems
for the synthesis of the two types of nucleic acid.

D. Conclusions

It is true that the study of the distribution of enzymes in the cell has only
just begun, nevertheless they have brought to our notice several important
facts concerning cellular metabolism. From these studies we see that
the cytoplasm is the principal region for the performance of anaerobic
degradations which provide the mitochondrial machine with fatty acids,
amino acids, and pyruvic acid. Since most of the glycolysis occurs in it,
the cytoplasm is manufacturing energy-rich bonds by an anaerobic pathway.
In addition the cytoplasm, or more particularly the microsomes, appears
to be the home of protein synthesis.

In the mitochondria is localized the respiratory part of the priming
reactions, the fatty acid cycle and the tricarboxylic acid cycle, the chief
providers of the ATP required for cellular work. However it appears that
the transfer of electrons from DPNH or TPNH to cytochrome-c can also
take place apart from the mitochondria.

With its enzymes for the synthesis of nucleosides and nucleoproteins,
the nucleus appears to be the repository of the specific DNA structure
and the apparatus for its transmission. It also has a part to play in the
control of the synthesis of the cytoplasmic nucleotides and of protein
synthesis in the cytoplasm. Whether the latter is controlled through the
synthesis of the microsomes or otherwise, the part played by the nucleus
has been demonstrated on numerous occasions although the mechanism
still remains to be discovered.

REFERENCES

Allfrey, V. G., MiRSKY, A. E. and Stern, H. (1955). The chemistry of the cell
nucleus. Advanc. EnzymoL, 16, 411-500.

CELLULAR TOPOCHEMISTRY 281

Claude, Albert (1947-48). Studies on cells : morphology, chemical constitution,

and distribution of biochemical functions. Harvey Lectures, 1950, 43, 121-164.
DouNCE, A. L. (1950). Cytochemical fundations of enzyme chemistry. The Enzymes.

Chemistry and Mechanism of Action. Sumner, J. B. and Myrback, K. (Editors)

vol. I, Part 1. Academic Press, New York. 187-266.
DE DuvE, Chr. and Berthet, S. (1954). The use of differential centrifugation in

the study of tissue enzymes. Internat. Rev. CytoL, 3, 225-275.
HooGEBOOM, G. H. and Kuff, E. L. (1955). Relation between cell structure and

cell chemistry. Fed. Proc, 14, 633-638.

CHAPTER II

CELLULAR REGULATION

The complexity of the priming reactions and the reactions which follow,
and their interrelations, lead us to ask how the direction of the molecules
along the various metabolic paths is controlled. Not only do the directions of
these metabolic reactions, all of which are proceeding at the same time, have
to be controlled, but also the velocities of these reactions must be regulated.
For example, how is it that in a cell liberally provided with substrates and
oxygen, respiration during a given period is no greater than the production
of utilizable energy ? These are a few of the questions we have to answer
when considering the ways in which the metabolism of the cell is regulated,

I. FACTORS WHICH DETERMINE THE VELOCITY
AND THE PATH OF ENZYMATIC REACTION CHAINS

By "reaction chain" the biochemist means a process made up of a series
of chemical reactions joined together in a straight or in a branching manner.
The idea that in a chain of reactions it is the slowest reaction which deter-
mines the overall speed of reaction has often appeared to be self-evident.
Hinshelwood illustrates this concept by comparison with the transmission
of telegraph messages. The speed of transmission depends upon a certain
number of factors — the dexterity of the operator, the speed of the current
along the wires, and the rapidity with which the telegraph boy delivers the
message. It is undoubtedly, says Hinshelwood, the last factor which is
the dominating one. This is no doubt true, but a series of biochemical
reactions differs from the sending of a telegram. As pointed out by Burton,
the agility of the telegraph boy does not normally depend on the number of
telegrams waiting at the post office, whilst the velocity of biochemical
reactions is influenced by the concentrations of the reactants, according
to the law of mass action.

The stationary state for a biochemical reaction chain may perhaps be
better compared. Burton points out, to that of the current in a stream. If an
obstacle is placed across the stream, there will be a temporary decrease in
the amount of water reaching the river mouth, but as soon as the water
level has reached the top of the dam, a new steady state will establish itself
and in the last analysis the amount of water arriving at the mouth of the
stream will depend upon only one factor, the amount of rain falling at the

282

CELLULAR REGULATION 283

source, which surely cannot be considered as the slowest process present.
A series of different factors can influence the resuhant of a number of
reactions catalysed by a system of enzymes, both their velocity and the
direction at alternative pathways and intersections will be affected.

A. Enzyme Concentration

In a chain of biochemical reactions, the concentration of an enzyme
affects the velocity of the metabolic reaction it catalyses. The relative
importance of two divergent paths at a fork in the metabolic chain can
therefore be influenced by the concentrations of the participating enzymes.
However, it is difficult to know with certainty what is the active concen-
tration of a given enzyme and there are only a very few measurements of
this type.

B. Kinetic Characteristics

The velocity of an enzyme reaction is defined as the change in substrate
concentration per minute. The velocity of the reaction depends not only
on the concentration of the enzyme, but also on the turnover number.

v=W.Ce
V = velocity of the W = turnover number Ce = enzyme

reaction (in moles of (in moles of substrate/ concentration

substrate/1, per min). min per mole of enzyme). (moles/1.)

Under conditions where the enzyme is saturated with substrate, the
turnover number is defined as the number of substrate molecules acted upon
per minute by one mole of enzyme.

The turnover number and the enzyme concentration (see A, above) are
therefore to be considered separately as regulating factors rather than their
product which is the reaction velocity. However, conditions where the
enzyme is saturated with substrate are rarely realized in cells and under
actual conditions the Michaelis constant K^i is more useful since it
enables us to calculate the reaction velocity for each substrate concentration.

C. Relation Between Thermodynamic Equilibrium
AND THE Stationary State

In the case of a reversible reaction situated in the middle of a chain of
reactions, when the stationary state is set up the establishment of
equilibrium is only possible if the velocities of the reactions situated on
either side of the reversible reaction are sufficiently low. If they are faster
than the reaction in the middle, equilibrium will not be attained (see
p. 148). There are cases where thermodynamic equilibrium is attained and
where the concentrations of certain products (which, under these
conditions, depend on equilibrium constants) decide the subsequent course

284 UNITY AND DIVERSITY IN BIOCHEMISTRY

of metabolism. Such a case are the stages of glycolysis between phospho-
glyceraldehyde and 3-phosphoglyceric acid in the course of which an
energy-rich bond is formed (p. 189). 1,3-Diphosphoglyceric acid undergoes
a spontaneous hydrolysis into 3-phosphoglyceric acid and inorganic
phosphate. The equilibrium constant for the enzymic conversion of
phosphoglyceraldehyde into 1,3 -dip hosphogly eerie acid is such that only a
small amount of this acid is present at equilibrium. For the step from
1,3 -dip hosphogly eerie acid to 3-phosphoglyceric acid, with the formation
of ATP, the equilibrium constant favours the formation of a relatively large
amount of the latter. When the two reactions are combined, a stationary
state is set up in which the concentration of 1,3-diphosphoglyceric acid is
very small. In this way the slow spontaneous hydrolysis of the latter is kept
to a point where it is insignificant,

D. The Concentration of Coenzymes

An example of the importance of this in the regulation of metabolic
processes is provided by the antagonistic effect of K+ and Na+ ions on the
second transfer of phosphate during fermentation.

E. Temperature

Since temperature does not affect all enzymes in the same manner its
influence is such as to cause changes in biochemical reactions.

F. Permeability of the Outer Region of the Cytoplasm

This acts as a regulator of metabolism by controlling the passage of
substrates or metabolites. An example of the first mode of control is the
fact that yeast ferments glucose but does not ferment F-1,6-PP, to which
the surface of the cell is impermeable.

The second type of effect is illustrated by the ready permeability of the
outer region of the yeast cell to ethyl alcohol. But for this ready perme-
ability, the accumulation of alcohol inside the cell would soon cause
alcoholic fermentation to cease.

G. Topobiochemistry

The cellular topobiochemistry of enzymes, as it has been described in
the preceding chapter, offers numerous possibilities for intracellular
regulation of reactions; yet the study of this subject has hardly begun.
During alcoholic fermentation, for example, ATP is formed in the cyto-
plasm and transferred to cellular particles containing ATP-ase, from
whence the molecules return to the cytoplasm to replenish the stock of
adenine nucleotides acting as phosphate acceptors (AMP, ADP). The
diffusion of ATP away from the cytoplasm and the diffusion of AMP and
ADP towards the cytoplasm evidently form one of the regulating processes
in glycolysis.

CELLULAR REGULATION 285

During mitochondrial respiration, on the other hand, the turnover of
phosphate in the adenine nucleotides is more rapid than the diffusion of
ATP to the outside of the mitochondria or of phosphate acceptors to the
inside.

But the concentrations of AMP, ADP and ATP remain constant in the
stationary state in mitochondria during active oxidative phosphorylation.
So apparently there is a control of oxidative phosphorylation by means of a
regulation of the entry of nucleotide phosphate acceptors. According to
Siekevitz and Potter, this entry is under the control of an enzyme situated
in the outer region of the mitochondria, this enzyme, adenylic kinase,
catalyses the establishment of equilibrium between AMP, ADP and ATP.

H. Stationary States

The intervention of the various factors responsible for intracellular
regulation leads to the persistence over an extended period of concen-
trations of the various substances which compose the cell. The stationary
states themselves also serve to regulate the orientation of a reaction chain
where alternative pathways are possible.

II. THE PASTEUR EFFECT

From the results of experiment it has been possible to deduce that the
control of cellular oxidation depends upon two factors : the concentration
of inorganic phosphate and the concentration of phosphate acceptors. An
example of the regulatory influence of these concentrations is the so-called
Pasteur Effect. First observed by Pasteur, as indicated by the name, this
phenomenon consists of the fact that when a cell is using oxygen glycolysis
proceeds less rapidly than in the absence of oxygen. Although glycolysis
is not a process in which oxygen actually plays a part, yet it is partially
inhibited by the presence of oxygen. In a cell lacking oxygen a much greater
number of sugar molecules undergo glycolysis than in the presence of
oxygen. Yet, in the second case, the amount of useful energy, obtained in
the form of energy-rich bonds, is much greater. From a fundamental point
of view the Pasteur Effect is of great interest, but it is its mechanism, as an
example of intracellular regulation, which interests us here.

The Pasteur Effect can be described by saying that "aerobic glycolysis"
is weaker than "anaerobic glycolysis". Also, in cells during growth and in
cancer cells, aerobic glycolysis is greater than in resting cells, hence the
statement of Warburg; "No growth without glycolysis". The explanation
of the Pasteur Effect has been provided by Lynen who has shown that the
intensity of glycolysis depends on the concentration of inorganic phosphate
available at the stage of the dehydrogenation of phosphoglyceraldehyde
with production of 1,3-diphosphoglyceric acid.

When the oxidative phosphorylations of respiration take place, the

286 UNITY AND DIVERSITY IN BIOCHEMISTRY

resulting consumption of inorganic phosphate brings about a decrease in
the intensity of glycolysis, and inversely. The Pasteur Effect is therefore an
example of an autoregulation of all the complex phenomena of the priming
reactions. Another factor in the regulation of the intensity of respiratory
metabolism is operative in the mitochondria, and this is the supply to the
interior of the mitochondria of phosphate acceptors such as AMP and ADP.
To the autoregulatory processes of this type can be added the action of
specialized regulators such as the hormones which have been developed
by organisms in the course of biochemical evolution.

III. THE GENETIC CONTROL OF THE RELATIVE
RATES OF ENZYMATIC REACTIONS

Although it is true that the cells of various organisms all possess meta-
bolic systems having the general features described in part three of this
book, each species has certain specific peculiarities in the macromolecules
of which it is formed, the enzymes included.

Heredity transmits to the descendants of an organism the specific type
of control of the relative rates of the diverse enzymic reactions which take
place in each of its cells. It has now been well established that an alteration
in a given gene can bring about definite biochemical changes manifested
by the disappearance of one constituent in the organism, or the appearance
of a new one, or by an increase or a decrease in the amount of a compound
or of a group of compounds. New stationary states are set up resulting from,
changes in the speed of this or that metabolic process. For example, in a
given organism consider the concentration of a substance A. In the station-
ary state in the organism, the concentration of A remains approximately
constant as a result of an equilibrium between the production process and
its transformation. If the rate of production decreases, even slightly, and if
the rate of transformation does not alter, then substance A will disappear.

Although the biochemical phenotype, like phenotypes in general, is a
product of the interaction of the internal milieu of the cell, and its genotype,
the means by which the latter influences the system of macromolecules in
the cell and in particular the enzymes still remains a mystery.

REFERENCES
HoLZER (1953). Tiber Fermentketten und ihre Bedeutung fiir die Regulation des

KohlenhydratstoflFwechsels in lebenden Zellen. Biologic und Wirkung der

Fermente. 4. Colloquium der Ges. fiir physiol. Chem., Springer, Berlin-

Gottingen-Heidelberg. 89-112.
Lardy, H. A. (1952). The role of phosphate in metabolic control mechanisms.

The Biology of Phosphorus, Wolterink, L. F. (Editor) Michigan State College

Press, 131-147.
Potter, Van R. (1949). The control of metabolism. Respiratory Enzymes, L,ardy^

H. A. (Editor) Burgess, Minneapolis, revised ed., 264—272.

PART FIVE
BIOCHEMICAL DIVERSITY

4

CHAPTER I

SOME ASPECTS OF BIOCHEMICAL DIVERSITY

The chemical processes described in Part Three of this book give an
approximate and overall view of the metabolism and biosynthetic mechan-
isms in cells. However, numerous variations on these themes are possible
and a few examples follow.

I. TERPENES

We have described (p. 235) the biosynthesis of isoprene from acetyl-CoA
as it usually occurs in cells. In the essential oils of plants we find a large
number of compounds which demonstrate the large number of possible
compounds which can be formed in a similar manner, starting from acetyl-
CoA. They are compounds made up of isopentane units. They contain 5,
10, 15, 20 or more carbon atoms and are called respectively, hemiterpenes,
mono-, sesqui-, di- or polyterpenes. From the material which is not distill-
able in steam, by solvent extraction it is possible to obtain a series of other
substances containing 20, 30, 40 carbon atoms or more and belonging to
the groups of diterpenes (i.e. the resins), the triterpenes (i.e. the saponins),
the tetraterpenes (i.e. the carotenoids) or to the polyterpenes (i.e. rubber).
Moreover a whole series of organic compounds synthesized by plants are
related to isopentane since they contain such units in their structure.
Among these isoprenoids are the irones. There are many monoterpenes in
plants and in general, but not always, one can consider their formula as
being based on two isopentane units joined in head to tail union. The
sesquiterpenes can be considered as formed from three isoprene units in
head to tail union. The cyclic monoterpenes and sesquiterpenes can be
considered as resulting from the rolling up of the same chains.

Certain of the diterpenes can be considered as containing four wopentane
units in head to tail union. This is the case of phytol and vitamin A. Others
have an irregular arrangement.

Among the tetraterpenes, those related to lycopene and called caroten-
oids have been described previously). Plants are able to synthesize
carotenoid molecules whilst animals are only able to modify them, for
example by oxidation. Astaxanthin a carotenoid usually found in
crustaceans, is one such oxidation product.

In mammals, birds, and certain amphibians, the ingestion of carotenoids in
the food results in an absorption of carotene in the intestine, the extent of

289 u

290 UNITY AND DIVERSITY IN BIOCHEMISTRY

absorption depending on the greater or lesser activity of the intestinal caro-
tenase which converts carotene to vitamin A and as a result the reserve fats be-
come more or less saturated w^ith carotene. Man and other Primates absorb
carotenoids in general, as does the frog also ; other animals exercise a selective
adsorption. For example, the horse and the cow selectively absorb caro-
tenoids and store them without alteration, whilst birds and fish show a pre-
ference for xanthophylls. However birds and fish modify one of the ingested
xanthophylls, lutein, by oxidation, and the products of the oxidation are
deposited in the feathers in the case of birds, and in the skin in the case of fish.

The carotenoid structure appears to be connected in a general way with
the function of photoreception. The most primitive type of photoreception,
lacking the presence of any differentiated photoreceptors, is the type called
dermatoptic, which is found in primitive types of organisms up to the
amphibians and also in plants. The maximum sensitivity of this derma-
toptic function is in the ultra-violet part of the spectrum around 365m/u-,
and it is the receptor in photokinetic processes involving tropisms towards
light. Now, in a number of cases, photoreceptors have evolved secondarily
and developed new kinds of receptor molecules adapted to the light from
the sun and the sky. All these substances belong to the carotenoid group.
In plants, phototropic bending depends on the properties of carotenoids
such as xanthophyll in Avena, or /3-carotene in the sporangiophores of
Phycomyces. The orientation of an animal depends on visual photoreception
and requires the presence of other carotenoids showing the same kind
of adaptation to sunlight and having an absorption maximum at around
500m/Li. This development is due to the ability, mentioned above, to change
some of the plant carotenoids into vitamin A. There are two types of
vitamin A : vitamin A^ and vitamin Ag.

Let us first consider the system in the eye of land vertebrates such as
mammals or birds. The pigment of their retina is rhodospin, a rose-
coloured carotenoid-protein complex. In aqueous solution its absorption
spectrum consists of a single broad band with a maximum at 500m/x. In
light it is bleached to orange and yellow pigments and in the process the
carotenoid retinene I is liberated. The latter substance has never been
found anywhere except in the retina. Its spectrum in chloroform consists of
a single band with a maximum at about 387m)Lt. In the retina the mxture of
retinene I and protein reverts to rhodopsin and in addition retinene I is
converted to vitamin Ai, which, in the intact eye, also reunites with protein
to form rhodopsin. This system is not only present in the eyes of mammals
and birds but also in the eyes of Invertebrates such as the squid, Loligo,
and the crayfish Camharus.

If we consider the system in the eye of a marine fish we find the rhodopsin
system as in birds and mammals and Invertebrates, but this is not the system
to be found in the eyes of freshwater fish which contain another system,

SOME ASPECTS OF BIOCHEMICAL DIVERSITY

291

the porphyropsin system. Porphyropsin, like rhodopsin, is a carotenoid-
protein complex and is purple in colour. Its spectrum resembles that of
rhodopsin, but with a maximum at 522m/^. On exposure to light a sub-
stance having properties similar to rhodopsin is liberated; it is called
retinene II. In chloroform it has an absorption maximum at 405m/x. In the
retina it is converted simultaneously to porphyropsin and to vitamin Ag.

II. PORPHYRINS

We have described the biosynthesis (p. 238) of porphyrins from 8-
aminole\ailinic acid via porphobilinogen. On to this basic process are
superimposed a considerable number of variations. By insertion of iron into
the protoporphyrin nucleus we obtain w^hat Granick has called the "iron
branch" of the biosynthetic chain (p. Ill), and a number of other variants
have been described in these pages. Protoporphyrin is also the starting
point for the biosynthesis of chlorophyll (the "magnesium branch" of
Granick) as well as for the biosynthesis of haem.

A cell capable of photosynthesis (see p. 354) contains at least one
chlorophyll and at least one yellow pigment. In addition it often contains a
phycobilin. The chief pigment in photos}^nthesis, both in algae and in the
higher plants, is chlorophyll a. In the photosynthetic bacteria, on the other
hand, we find a different chlorophyll, bacteriochlorophyll (p. 122). Thus
whilst in green plants the chloroplasts contain chlorophyll a and chloro-
phyll b, the algae are much more variable and we find in them a number of
combinations .a -\- b, a -\- c, a -\- d, a + e. In addition we sometimes find
a phycobilin. The phycobilins are soluble in water and are proteins com-
bined with a chromophore belonging to the class of bile pigments. The
phycoerythrins are predominant in the red algae and the phycocyanins in
the blue-green algae.

The chromophore of the phycoerythrins, phycoerythrobihn, is identical
wuth mesobilierythrin the formula of which is

ME MP P M ME

HO H

CH, H

CH

N

CH N OH

(M = methyl group E = ethyl group P = propyl group)
The chromophore of the phycocyanins is mesobiliviolin

MEMP PMME

/\ /\C/\N/\ /\ /\C//\N\\
HO N H H CH

N

H,

H OH

Various phycoerythrins are found in algae which differ in the structure of
the protein moiety. 2-Phycoerythrin is the most common and is found in the

292 UNITY AND DIVERSITY IN BIOCHEMISTRY

Rhodophyceae whilst c-phycoerythrin is present in the Myxophyceae.
Among the phycocyanins, r-phycocyanin is present in the Rhodophyceae
and c-phycocyanin in the Myxophyceae. The phycobiHns serve to absorb
light and transmit energy to other systems, notably the chlorophyll system.

Plants are able to continue the synthesis of porphyrins along the "iron
branch" and along the "magnesium branch" whilst in animals the latter is
lacking. However, animals have particularly developed the "iron branch"
as far as the biosynthesis of the compound of haem and globin, haemo-
globin, is concerned. The biosynthesis of haemoglobin is sometimes
observed in plants, for example in the root nodules of Legumes. In animals,
the presence of haemoglobin in tissues other than blood has often been
demonstrated. For example, it has been shown to occur in the nervous
system of certain worms e.g. in the Annelid Aphrodita and in a number of
the Nemertea : Polia, Meckelia and Borlatia.

In Insects haemoglobin is found in the Diptera and the Hemiptera.
In Gastrophilus intestinalis which, during its larval period, is a parasite in
the stomach of the horse, the young larva is a uniform red due to the
coloration, by haemoglobin, of the fatty bodies, of the parietal muscles and
of the hypodermis. As the larva grows further the haemoglobin becomes
localized in special cells, the tracheal cells, forming a red mass localized in
the posterior third of the body.

In certain of the Hemiptera such as Buenoa margaritacea and Anisops
producta, the haemoglobin is similarly localized in masses made up of
tracheal cells. In addition in another Hemiptera, Macrocrixa geoffroyi,
haemoglobin is present in the accessory glands of the male genital system.

Vertebrate muscle contains a haemoglobin known as myoglobin and the
respiratory pigment has also been demonstrated in the pharyngeal muscles
of a number of gastropod molluscs {Pahidina, Littorina, Limnaea, Patella,
Chiton, Aplysia) and also in the body wall of Ascaris lumbricoides.

The presence of haemoglobin in the blood is a general characteristic of Ver-
tebrates: it is always contained in blood cells, either nucleated or non-
nucleated. Nevertheless, some fishes adapted to cold waters have been shown
to have no haemoglobin and no er)^hrocytes in their blood whatsoever.

The distribution of haemoglobin in the blood of Invertebrates, where it may
be present either in cells or dissolved in the blood, defies all systematization.

Among the Echinoderms, corpuscles containing haemoglobin have been
demonstrated in one, sometimes in two, and occasionally in all three of the
body fluids of the Ophiuroidea and in several of the Holothuroidea.
Corpuscles have never been found in the Asteroidea, the Echinoidea, nor in
those Holothuroidea that possess a test.

The presence of a haemoglobin in the blood or coelomic fluid of annelid
worms has long been known. In the Polychetes there is generally a cir-
culatory system and a red blood containing dissolved haemoglobin

SOME ASPECTS OF BIOCHEMICAL DIVERSITY 293

(Arenicola, Eunice, Cirrhatulus, Nephthys, Nereis, Nais, Ophelia, Marphysa,
etc.). In the Phyllodocidae, Syllidae and Chaetopteridae, the blood is
colourless. No case is known of an Annelid having a blood containing an
intracellular oxygen-carrier. Where there is haemoglobin in the coelomic
fluid of an Annelid, the oxygen-carrier is always contained in a blood
corpuscle. This is the case for a number of Polychetes not possessing a
circulatory system, e.g. the Capitellidae, Glyceridae and Terebellidae
Polycirrus hematodes. In other Terebellidae such as Travisia forbesii or
Terebella lapidaria, there is also dissolved haemoglobin in the coelomic
corpuscles. Among the Oligochaeta, we generally find colourless blood
in the circulatory system of the Enchytraeides (though some of them,
such as Pachydrilus lineatus, have haemoglobin in their blood). In many
others of the Oligochaeta it contains dissolved haemoglobin: in Lumbricus,
Tubifex, Limnodrilus, Lumbriculus, etc. When we come to the Hirudinea,
we find dissolved haemoglobin in the blood of the Gnathobdellides
(Hirudo, Aulastoma, Nephelis, etc.) and colourless blood in the Rhynchob-
dellides [Pontobdella muricata, Branchiobdella astaci, etc.).

In the nemertian or the turbellarian worms, we sometimes find haemo-
globin-containing corpuscles in the blood [Derostoma, Syndesmis, Dre-
panophorus, Polio).

The Echiurioidea are sometimes considered as aberrant worms. They
are not segmented and only possess a few chaetae arranged differently from
that in the Annelids and their body consists of two parts, a retractile preoral
lobe and the body proper. In the coelomic cavity of many Echiurioidea
nucleated corpuscles containing haemoglobin are found. Moreover the
coelomic fluid of the Echiurioidea is the only fluid in their milieu interieur
for they are devoid of a circulatory system.

Among the molluscs, we find haemoglobin-containing corpuscles in the
blood of numerous Lammellibranchs (belonging to the genera Pectunculus,
Glycimeris, Cutellus, Area, Gastrana, Tellina, Solen, Poromya, Capsa,
Astarte, etc.) and in the coelomic fluid of certain Amphineura, the Neomen-
ians. In a Gastropod, Planorbis, the blood contains dissolved haemoglobin.

Only very exceptionally do we find haemoglobin in the Arthropods.
Among the Crustacea, many species of small size are without an oxygen
carrier whilst others have haemocyanin in the blood. It is only in the group
of the Entomostracea [Apus, Branchipus, Artemia, Daphnia, Chirocephalus,
Lernanthropus, Clavella, Congericola) that we find bloods containing dis-
solved haemoglobin. When we come to the insects, who, in order to bring
oxygen to the cells, have had recourse to a direct transfer from the air via the
tracheae, we find that their blood is without an oxygen carrier, with the re-
markable exception of the blood of the larvae of certain of the Chironomides.
The similarity to haemoglobin of the red pigment dissolved in the blood
of the larva of the Diptera Chironomus, was demonstrated by Rollet in 1861.

294

UNITY AND DIVERSITY IN BIOCHEMISTRY

The term haemoglobin applied to certain molecules implies that they
have a number of common characteristics and among them :

(a) that they are derived from protohaem; the prosthetic groups of
different haemoglobins when combined with a given basic nitrogen com-
pound, in general give rise to the same haemochromogen.

(b) that of being heteroprotein in nature, the protohaem being united to
a holoprotein to which the name globin has been given ;

(c) that of giving a characteristic spectrum, two-banded in the oxygen-
ated state and one-banded in the reduced state.

{d) that of complexing reversibly with molecular oxygen instead of being
oxidized by it to a ferrihaemoglobin. (As previously stated (p. 117) this
property is due to the globin forming a paramagnetic complex with the
ferroporphyrin, whilst the other haemochromogens are diamagnetic).

(e) that of combining with carbon monoxide to give carboxyhaemo-
globin, whose visible spectrum has two bands in positions different from
those of the corresponding oxyhaemoglobin ;

(/) that of being, when oxygenated and in pure solution, transformed
more or less slowly into methaemoglobin, containing trivalent iron and no
longer capable of being oxygenated, the solution of this compound giving
an absorption band in the visible region differing from that given by
reduced haemoglobin.

The relationship implied by use of the term haemoglobin applied to
haem-proteins which can be oxygenated and contain the base protohaem,
does not preclude them possessing differences which is revealed when a
comparison is made.

Besides the fact that haemoglobin crystals differ from one animal species
to another, the amino acid composition of different haemoglobins show
clear-cut differences as the examples in Table XIV indicate.

Table XIV

(Roche and Jean; Roche and Mourgue)

Mean amino acid composition of various haemoglobins

Trypto-

Tyro-

Cys-

Argi-

Histi-

Ly-

Leu-

Val-

Ala-

Haemoglobin

phane

sine

tine

nme

dine

sine

cine

line

nine

of

%

%

0/

/o

0/

/o

0/

/o

0/
/O

0/

/o

0/

/o

%

Lumbricus

4-41

3-47

1-41

10-07

4-68

1-73

Arenicola

1-64

2-52

4-08

1004

4-03

1-85

9.688

6-69

—

Horse

2-38

3-38

0-74

3-57

8-13

8-31

1915

9-92

8.93

The haemoglobin of Vertebrates is clearly different from other haemo-
globins in containing less arginine and cystine and more histidine, lysine
and leucine.

SOME ASPECTS OF BIOCHEMICAL DIVERSITY

295

The positions of the a and ^ bands in the spectrum of the oxyhaemo-
globins are also different : in the AnneHds the two bands are shifted
towards the violet end of the spectrum relative to the positions in the
Vertebrates. Conversely, in the Holothuria the two bands are shifted
towards the red end.

The isoelectric points of the various haemoglobins also show differences
(see Table XV).

Table XV

Isoelectric points of haemoglobins

(see Florkin, 1948)

Planorbis

4-77

Thyone briareiis

5-80

Nereis virens

5-10

Arenicols marina

4-76

Aphrodite aculeata

5-70

Glycera convoliita

5-60

Haetnopis sanguisuga

5-01

Liimbricus terrestris

5-28

Chironomus plumosiis

5-40

Gastrophilus intestinalis

6-20

Man

6-78

Horse

6-78

Pigeon

7-23

As can be seen from the Table, vertebrate haemoglobin has an iso-
electric point around neutrality and differing from that of other types of
haemoglobin whose isoelectric points are more acid.

The diversity' in the molecular weights of a series of haemoglobins is
illustrated by Table XVI.

Table XVI
The molecular weights of haemoglobins

(see Wyman, 1948)

Based on sedimentation

Basec

on equilibrium

constant S20

studies

in ultracentrifuge

Haemoglobins

Thyone

23,600

Arenicola

3,000,000

Notomastus

36,400

Liimbricus

3,150,000

2,950,000

Planorbis corneus

1,630,000

1,540,000

Area

17,100

19,100

Myxine

23,100

Chironomus

31,500

Man

63,000

Horse

68,000

68,000

Cytochrome-c

15,600

13,000

Myoglobin (horse)

16,900

17,500

296 UNITY AND DIVERSITY IN BIOCHEMISTRY

The behaviour of the various haemoglobins in the course of oxygenation
gives us a further means of characterizing them. This is done by plotting
a graph showing the degree of oxygenation as a function of the partial
pressure of oxygen and is commonly known as the dissociation curve of
oxyhaemoglobin. If we represent by T the portion of the haemoglobin
molecule corresponding to a group capable of being oxygenated, i.e. an
iron atom, then the equilibrium will be represented by

TO^^T + Oa (1)

K being the equilibrium constant for the oxygenation. Since the concen-
tration of oxygen in the solution, [Og], is proportional to the partial pressure
of oxygen in accordance with Henry's Law, we can replace it in equation (2)
by the partial pressure p.

If we represent the concentration of oxygenated haemoglobin by
[HbO,^ and that of the non-oxygenated haemoglobin by [Hh\ the equation
becomes

log i-j^-" = log ^ + log K (4)

The logarithmic form is particularly useful since if we plot the values of
log [//6O2] / [//6] as ordinates and the values of log p as abscissae, then
since i^ is a constant, we shall obtain a straight line inclined at 45° and
cutting the ordinate axis at the value of log K. In the usual form where the
percentage saturation is plotted on the y axis against partial pressures on
the X axis, one obtains a hyperbola. This is the case for the muscle haemo-
globin of mammals and the larval haemoglobin of Gastrophilus. In the most
general case the dissociation curve is not a hyperbola but a sigmoid-shaped
curve whose form can be empirically expressed by the well-known
equation :

[HbO;\ ,^
[Hb] ^

in which the exponent represents, although rather vaguely, the degree of
interdependance or difference of the various groups which are oxygenated.
In fact, in solutions of the muscle haemoglobin of Vertebrates and the
haemoglobin of Gastrophilus there is only one type of oxygen-binding
group. Myoglobin contains only one iron atom, and the larval haemoglobin
contains two atoms which are identical in character. In haemoglobins
giving a sigmoid curve, either the oxygen-binding groups in the same

SOME ASPECTS OF BIOCHEMICAL DIVERSITY

297

molecule have different oxygenation constants or the oxygenation of one
modifies the constants of its neighbours. In general, the dissociation curves
of Vertebrate haemoglobins have a sigmoid form but the degree of flatten-
ing varies from one class to another (Fig. 86).

Fig. 85 (Keilin and Wang) — Dissociation curve of a concentrated solution (1 X 10~' g-
mols haematin per litre) of a Gastrophilus haemoglobin. Temperature 39°C.

One can obtain an idea of the affinity of a particular haemoglobin for
oxygen by noting the position of its dissociation curve at the p^Q value, i.e.
the partial pressure of oxygen corresponding to 50% oxygenation.

However it is necessary to compare values oi p^o obtained under com-
parable conditions. Changes in temperature and pH displace the dissocia-
tion curve. The combination of molecular oxygen with a carrier being an
exothermic process, an increase in temperature will lower the affinity, and
a decrease in temperature will increase it. Figure 87 illustrates the shift
in the dissociation curve of the oxyhaemoglobin of the ray Rata ocellata
with temperature.

The influence which changes in the partial pressure of carbon dioxide
(pco,) have on the affinity of oxygen for haemoglobin is known as the

298

UNITY AND DIVERSITY IN BIOCHEMISTRY

"Bohr effect". This influence of changes in the Pqq^ is due to changes in
the pH causing changes in the dissociation of the amphoteric haemoglobin
and thus ahering the properties of the oxygen -binding groups. When the
Pqq^ of an alkaline solution of haemoglobin is increased, the pH approaches

100

Fig. 86 (Morgan and Chichester) — Dissociation curves of haemoglobins of man,

dog and pig.

the isoelectric point of the carrier and the dissociation of its acidic groups
diminishes. The affinity of haemoglobin for oxygen diminishes likewise
and the p^o value increases since the dissociation curve is displaced to the
right. Figure 88 illustrates this phenomenon for horse haemoglobin.
Since the influence and extent of the Bohr effect is very variable, to compare
the oxygen affinities of different haemoglobins it is necessary to compare

SOME ASPECTS OF BIOCHEMICAL DIVERSITY

299

0 20 40 60 80 100 120 140 160 180 200 220 240 260

pOg

Fig. 87 (Dill, Edwards and Florkin) — Dissociation curves of haemoglobin of Raia ocellata

at diflferent temperatures. >

V ■■«*'■■

Fig. 88 (Ferry and Green) — Influence of changes in pH on the dissociation curve of

horse haemoglobin.

300

UNITY AND DIVERSITY IN BIOCHEMISTRY

dissociation curves obtained under similar conditions of pH and tempera-
ture. These conditions have not yet been satisfactorily fulfilled, since a
change in the ^^Oa alters the pH of the blood and the pH of a solution of
haemoglobin in different ways, and the dissociation curve in each case is
displaced in a particular manner depending on the pH. The alteration in
affinity with temperature also differs, for solutions of haemoglobin, accord-
ing to the conditions and the nature of the haemoglobin. Whilst awaiting
more precise results it is still possible to arrive at some interesting con-
clusions using the results published in the literature.

Of the known haemoglobins the one with the greatest affinity for oxygen
is certainly that in the perienteric fluid of Ascaris lumbricoides. Ascaris
possesses two haemoglobins, one in the body wall and the other in the
perienteric fluid. These two haemoglobins differ from each other in their
absorption spectra, which are both different from that of pig haemoglobin.
The haemoglobins of Ascaris have a great affinity for oxygen : sodium
hydrosulphite only reduces them very slowly and exposure to vacuum at
20° does not reduce them completely.

The haemoglobin of Gastrophilus larva has a lower affinity for oxygen.
At 39° the ^50 of a concentrated solution containing 1 x 10~^g atoms of
iron per litre has a value of 4*9mm; whilst the ^50 of a dilute solution
containing 0"84 X 10~^g atoms of iron per litre is 0"02mm Hg. The further
complication we encounter here, namely the change in affinity with the
degree of dilution, also occurs with the different mammalian haemoglobins.

Fox has compared the various values of /)5o, at 10° and 17°, and in the
absence of COg, for the undiluted bloods of Chironomus larva, of Arenicola
and Planorbis, and for the slightly diluted bloods of Tubifex, Daphnia and
Ceriodaphnia. He obtained the results collected in Table XVII.

Table XVII

(H. M. Fox)

p50

10°

17°

Chironomus riparius
Tubifex sp.

Ceriodaphnia laticaudata
Arenicola marina
Planorbis corneus
Daphnia magna

0-5
0-5

1-5
1-5
20

0-6
0-6
0-8
1-8
1-9
3-1

In this list, the haemoglobins of the Chironomus and of Tubifex have the
highest affinity and that of Daphnia the lowest. We may compare the values
in the second column with the p^o value of 27mm for human blood with a

SOME ASPECTS OF BIOCHEMICAL DIVERSITY 301

normal haemoglobin concentration and at 20° and pH 7-4. It is clear that the
haemoglobin of Invertebrates has a distinctly higher affinity for oxygen
than human haemoglobin.

The various haemoglobins also differ with respect to the change in their
affinity for oxygen with pH (Bohr effect). Some haemoglobins do not show
a Bohr effect; e.g. the haemoglobins of Urechis, of ChiroTiomus larva and
vertebrate myoglobin. The haemoglobins of Invertebrates other than those
quoted above show only a very feeble effect {Ceriodaphnia laticaudata,
Thalassema neptuni, Arenicola marina, Planorbis corneas, Daphnia magna).
It is present in a more or less marked degree in the various classes of
Vertebrates.

The haemoglobins also differ from each other in other ways. If for each
haemoglobin we measure in angstrom units the span separating the position
in the absorption spectra of the a band of oxyhaemoglobin from the a band
of carboxyhaemoglobin, we find clear-cut differences.

In addition, the auto-oxidation of haemoglobin to methaemoglobin is
very slow for vertebrate haemoglobin and more rapid with that of Gastro-
philus, mammalian myoglobin, and the haemoglobin found in the roots of
legumes.

III. PROTEIN MACROMOLECULES

We have emphasized the specific character of macromolecules.
Even in the arsenal of enzymes common to all cells we find indications of
this specificity. The glucose dehydrogenase of vertebrate liver for example,
is not inhibited by toluene, whilst that of E. coli is inhibited. Yeast alcohol
dehydrogenase is completely inhibited by 0-001 M iodoacetate, whilst even
at a concentration of 0-OlM, animal alcohol dehydrogenase remains
unaffected. Glutamic acid dehydrogenase of yeast requires TPN as a
coenzyme whilst the same enzyme from plants needs DPN.

When examining the various characteristics of the haemoglobins we
noted a number of differences arising from the different properties of the
protein moiety.

Another aspect of diversity in macromolecules is provided by the pro-
duction, in animals, of protein molecules circulating in the blood in re-
sponse to the injection of various substances. These antibodies are specific
for the injection antigens (proteins, polysaccharides, etc.) and react vnth.
them to produce a precipitate, or agglutination if the antigen is attached to
a cell surface. The antibodies circulating in the blood are usually present in
the serum y-globulin fraction.

The antibody properties of these molecules is due to the special cha-
racteristics of a given part of the macromolecule and numerous facts
indicate that antibodies of various specificities are very similar to normal
globulin except at one very small part of the macromolecule. For example,

302 UNITY AND DIVERSITY IN BIOCHEMISTRY

it is possible to split the macromolecule of an antibody with a protease and
the antibody activit\^ will remain localized in one of the hydrolysis products.
Substances capable of stimulating the production of antibody are called
complete antigens and are generally protein in nature. However, a much
smaller molecule attached to the antigen molecule may be the site for the
specificity of action of the antibody so formed. This small molecule is known
as a haptene. An example of this t^'pe is the antigenic activity of ovalbumin
coupled to an azobenzoate ion. The antibodies formed will be of several
types, certain of them reacting with the protein part of the molecule of
antigen and others with its haptene, the azobenzoate ion. The antiserum
prepared from a rabbit would precipitate the ovalbumin combined with,
the azobenzoate, but not ovalbumin alone. On the other hand, the antibody
will combine with the free haptene but without giving rise to a precipitate.
But the molecules of antibody which are combined with the free haptene
will no longer combine with the antigen carrying the haptene and this
competition between antigen and haptene for the site on the antibody
molecule which is specific for the haptene will result in a decrease in the
amount of precipitate formed by the combination of the antibody and the
antigen.

The combination of the antigen with the antibody arises from the
complementary character of their structures, the two corresponding
regions of their molecules fitting each other in such a way that they are
bound together without the intervention of strong chemical bonds. The
complementary character of antigen and antibody is interpreted as due to
the antibody being formed by the folding of its protein chain against the
antigen acting as a template. According to Pauling the folding takes place so
that hydrogen bonds may be formed and that charge interactions can be
effective between the groups on the antigen and on the antibody.

Specific antiserums permit various antigens to be distinguished from
each other and consequently also the various types of organisms which
these antigens characterize.

Another aspect of diversity in marco molecules shows itself by the
insertion of the same enzyme unit into different systems.

The various systems containing the phenolase complex in animals and
plants (heterotypical aspects of the same enzyme) are described in an
excellent review by H. S. Mason (1955) and it will suffice to quote the
conclusion of this review :

"At the phylogenetic level of the plants, it appears to catalyze the for-
mation of intermediates in biosynthetic systems which produce the flower
pigments and related flavonoids, the lacs and lacquers, the simple and
polymeric tannins and their esters, the phenolic alkaloids, the quinones,
tropolones and simple plant melanins, and the lignins. At higher phylo-
genetic levels the phenolase complex catalyzes intermediate phases in the

SOME ASPECTS OF BIOCHEMICAL DIVERSITY 303

pigmentation of the teguments, feathers, scales, hair and eyes of the
chordates. A vital role for an enzyme of the phenolase type occurs during
the biosynthesis of adrenaline and noradrenaline, but this remains to be
clarified.

"Each of the numerous heterotypic expressions of the phenolase complex
is produced by a unique biochemical sequence which is characterized by
(1) a phenolase specificity becoming narrower with rise in the phylogenetic
scale, (2) a characteristic chemical position in a metabolic network, and
(3) a specific physical localization within cell and organ. These variables
give ample play to the 'chance combination of genes which results in the
development of short reaction chains utilizing substances whose synthesis
had been previously acquired' (Horowitz, 1945), and to the states and
composition of the environment which determine the extent to which the
inherited phenolase complex can carry out its primary reaction. In this
manner, the chemical structure of an enzyme and its substrate can be
expressed as one of a number of biological characters."

REFERENCES

Florkin, M. (1948). La biologic des hematinoproteides oxygenables. Experientia,

4, 176-191.
Goodwin, T. W. (1952). The Comparative Biochemistry of the Carotenoids, London,

Chapman & Hall.
Granick, S. (1954). Metabolism of heme and chlorophyll. Chemical Pathivays of

Metabolistn, edited by Greenberg, D. PvL, vol. II, New York, Academic

Press, 287-342.
Haagen-Smith, a. J. (1953). The biogenesis of terpenes. Ann. Rev. Plant Physiol.,

4, 305-324.
Mason, H. S. (1955). Comparative biochemistry of the phenolase complex.

Advanc. EnzymoL, 16, 105-184.
Pappenheimer, a. M. (Editor) (1953), The Nature and Significance of the Antibody

Response, Columbia University Press, New York.
Wyman, J., Jnr. (1948). Heme proteins. Advanc. Protein Chem., 4, 407-531.

CHAPTER II

THE INHERITANCE OF
BIOCHEMICAL CHARACTERISTICS

I. CONTROL OF BIOCHEMICAL CHARACTERISTICS BY GENES

Beadle has expressed in the following terms the generally held views today
on the transmission of biochemical characteristics :

"In order to exist as such, genes obviously must be capable of inducing
the formation of exact copies of themselves. ... In addition to catalysing
formation of more units like themselves, genes in general have hetero-
catalytic properties, that is they catalyse the formation of other substances.
... In determining the specific chemical and perhaps physical configuration
of protein molecules, genes directly determine enzyme specificities and
thereby control in a primary way enzymatic synthesis and other chemical
reactions in the organism. ..."

The colour of the fruit of the tomato Lycopersicon esculentum results from
the presence of a series of carotenoids whose existence depends on at least
three genes (T, R, B,). As far as the two genes T and R are concerned,
Table XVIII shows the carotenoid pattern in four pure strains of Lyco-
persicon of the four genotypes RT, Rt, rT and rt.

Table XVIII

The carotene content of four strains of tomato

(Mackinney and Jenkins, 1952)

Carotene type

Micrograms of carotenoid per gramme of fruit

RT
Red

Rt

Orange

rT

Yellow

rt
Intermediate

h\\-trans Lycopene

cis Isomers of lycopene

j3-carotene {dW-trans)

poly-cis-carotene

all-^raws-S-carotene

Phytofluene

70-130

5-10

0-0-1
3-5

40-55
3-12
8-15

20-15
4-7

0-0-5
1-3

Ca. 0-1

10-15
0-5-1-0

0-01
0-7-1-0

Total

80-150

75-150

3-7

15-20

304

THE INHERITANCE OF BIOCHEMICAL CHARACTERISTICS 305

The carotenoid concentration increases in the following order of
genotypes

rT rt Rt RT

and it can be seen that the chief result of substituting R for r is an increase
in the amount of carotenoid.

The flower pigments, which we described under the name anthocyanins
and whose glycones are called anthocyanidins, have been the object
of extensive genetic studies carried out at the John Innes Horticultural
Institute. The anthoxanthins by their presence can modify the colour of a
flower determined by the anthocyanins. A change in a single gene may
markedly alter the colour of a flower. For example, in the snapdragon
the yellow or white colour depends on particular combinations of the
same pair of allelomorphs, Y and y. A yellow colour corresponds to the
genot}'pes YY or Yy, whilst the recessive plants yy have white flowers.
The reader will find a number of descriptions of biochemical characteristics
controlled by genes in the work of Wagner and Mitchell (1955).

11. BIOCHEMICAL DIFFERENTIATION OF CELLS
IN A SINGLE ORGANISM

In the course of the ontogenesis of a multicellular organism, the various
cells, although characterized by having the same genot}^pe, may be different
biochemically. The process called "determination" by the embryologists
causes one or other set of genes to act, the result being apparent in the
differentiation of each class of cell, resulting from the influence of the
cytoplasm on the activity of the genes or on the products of genes. The
biochemical diversity of organisms is hence in fact a diversit}^ of cell
aggregates which are themselves biochemically differentiated.

The explanation of how cells coming from the same zygote are differen-
tiated biochemically, belongs to the domain of chemical embryology and
chemical genetics and is still largely conjectural.

In a remarkable series of studies on the content and history of the cell
theory, John R. Baker (1948) has provided a modern formulation of the
theory in the following seven propositions :

"I. Most organisms consist of a large number of microscopic bodies
called 'cells', which, in the less differentiated tissues, tend to be
polyhedral or nearly spherical.

"II. Cells have certain definable characters. These characters show that
cells (a) are all of essentially the same nature and (b) are units of
structure.

"III. Cells always arise, directly or indirectly, from pre-existing cells,
usually by binary fission.

306 UNITY AND DIVERSITY IN BIOCHEMISTRY

"IV. Cells sometimes become transformed into bodies no longer
possessing all the characteristics of a cell. Cells (together with these
transformed cells, if present) are the living parts of organisms : that is,
the parts which accomplish the synthesis of new material. Cellular
organisms consist of nothing except cells, transformed cells, and
material extruded by cells and by transformed cells (except that in
some cases water, with its dissolved substances, is taken directly from
the environment into the coelom or other intercellular space).
"V. Cells are to some extent individuals, and there are therefore two
grades of individuality in most organisms ; that of the cells, and that of
the organism as a whole.
"VI. Each cell of a many-celled organism corresponds in certain respects

to the whole body of a simple protist.
"VII. Many-celled plants and animals probably originated by the

adherence of protist individuals after division."
The fact that cells are all of essentially the same nature is described in the
first part of this book under the heading of the biochemical unity of organ-
isms, and this idea implies also for the biochemist the similarity of each cell
of a multicellular organism to a monocellular organism. The idea that
organisms are made up of cells and transformed cells, for the biochemist,
corresponds to the concept of the biochemical diversity of the cells of the
same organism, all having however the same genotype. To illustrate the
great variety of biochemical differentiation in cells having the same genotype,
we shall take as our example an adult mammal such as man. The cells of the
ectoderm become differentiated into the cells of the epidermis, of the glands
of the skin, of the adenohypophysis, into nerve cells and modified nerve
cells such as those of the neurohypophysis or the suprarenal medulla, etc.

To the biochemist, the epiderm cell in mammals is characterized par-
ticularly by the biosynthesis of keratin, a protein rich in S-S linkages. This
type of cell is also one of the sites of synthesis of cholesterol. The cells of
the sebaceous glands are notable in bringing about the rapid transformation
of the whole of their content into a mixture of many lipides, saturated
alcohols, and hydrocarbons, etc.

Among the large polyhedral cells of the adenohypophysis we find several
types : cells whose cytoplasm is only slightly chromophilic, acidophilic
cells or eosinophils (a cells), and basophilic cells (^ cells). The eosinophils
are differentiated in that they synthesize two hormones, somatotrophic
(growth) hormone, and the lactogenic hormone (luteotrophin). The basophi-
lic cells biosynthesize thyrotrophic hormone, adrenocorticotrophic hormone
(ACTH), follicle stimulating hormone and the luteinizing hormone. It was
once believed that the hormones of the anterior hypophysis were all proteins,
but this has been shown to be false for ACTH and is very doubtful where
the thryotrophic hormone is concerned.

THE INHERITANCE OF BIOCHEMICAL CHARACTERISTICS 307

The neurones or nerve cells are characterized biochemically by the
nature and concentration of the complex lipides they contain and by an
exceptionally high concentration of the enzymes of the priming reactions
which consume glucose, and notably of hexokinase. Also the concentration
of the a-decarboxylase of glutamic acid (coenzyme: pyridoxal-5 -phosphate)
is particularly high in neurones. The system for the amidation of glutamic
acid is also very abundant. The neurones are of two types. All the pregang-
lionic fibres of the autonomic nervous system are cholinergic as also are the
postganglionic fibres of the parasympathetic system and certain postgang-
lionic fibres of the sympathetic system (those serving the sweat glands
for instance). The motor nerves are also cholinergic. Besides the
quantitative aspect of acet^'lcholine synthesis, the cholinergic fibres are
characterized biochemically by the mechanism of the transport of this
substance along the fibre and its liberation at the extremities. The adrener-
gic nerv^es synthesize noradrenaline, apparently by decarbox}4ation of
dihydroxyphen^'lserine. The cells of the adrenal medulla synthesize
noradrenaline and adrenaline, the latter from phenylalanine as has been
proved by using phenylalanine marked with ^*C in the a-carbon, the
isotope later appearing in the a-carbon of adrenaline.

The argentaffin cells of the intestine are differentiated in that they can
synthesize 5-hydroxytryptamine, probably via 5-hydroxytryptophane.
Another biochemical differentiation of nervx cells is observed in the den-
dritic melanocytes which, in the skin and the eye, are specialized for the

HO

/\

-CHo — CHo — Nrin

NH

5 -hydroxy tryptamine

biosynthesis of m.elanin. By means of their t^Tosinase these cells transform
tyrosine to dihydroxyphenylalanine, or DOPA, a substance similar in
structure to noradrenaline. The cells of the neurohypophysis, another type
of specialized nerve cell, perform a special synthesis, that of vasopressin
and oxytocin.

The cells of the endoderm differentiate into the cells of the salivary
glands, the mucous cells of the digestive tract, the exocrine pancreas, the
hepatic parenchyma, the parathyroid, the insulin-producing tissue, the
thyroid and the pulmonary epithelium.

The cells of the mucous membrane of the digestive tract and its neigh-
bouring glands show many types of biochemical differentiation. The cells
of the salivary glands are either of the mucous type or of the serous type.

308 UNITY AND DIVERSITY IN BIOCHEMISTRY

These latter synthesize ptyalin, an endoamylase which is also an a-amy-
lase. When we come to the gastric mucosa, besides mucous cells we find
present parietal cells specialized for the secretion of hydrochloric acid by a
mechanism of active transport, and peptic cells which can synthesize
pepsinogen, from which an endopeptidase, pepsin, is formed.

The cells of the exocrine pancreas are remarkably specialized for the
production of a whole series of enzyme proteins. Among these proteins we
find a lipase, an amylase, a maltase, a lactase, invertases, an exopeptidase
(carboxypeptidase) and various proenzymes, notably trypsinogen and
chymotrypsinogen. These two latter substances are transformed in the
intestinal lumen into two endopeptidases, trypsin and chymotrypsin. The
secretory cells of the intestinal mucosa produce a series of exopeptidases :
leucine aminopeptidase, glycyl-glycine dipeptidase, aminotripeptidase,
glycyl-leucine dipeptidase, prolidase, etc. In addition they synthesize a
maltase, an invertase, an enterokinase, nucleotidases and nucleosidases.

One of the biochemical characteristics of the cells of the hepatic paren-
chyma, in the domain of carbohydrate metabolism, is the presence of
glucose-6-phosphatase. Its presence enables the liver cell to liberate glucose
from G-6-P. The liver cell is also able to set free glucose from G-l-P
and liver glycogen. The cells of the hepatic parenchyma are much more
active than other cells in utilizing CO2 for the synthesis of oxaloacetic acid
from pyruvic acid and of malic acid from pyruvic acid. The carbohydrate
metabolism of the liver cell is much more complex than that of other types
of cell for a multiplicity of operations is involved : conversion of glucose
into various hexoses, glycogenesis, glucose oxidation, synthesis of amino
and fatty acids, glycogenolysis and gluconeogenesis. In the degradation of
fatty acids, the liver cell is found to possess a special biochemical pecu-
liarity; there is a check on the speed of entry of acetyl- Co A into the tri-
carboxylic acid cycle. Even under normal conditions, the stationary state
of the cell in the hepatic parenchyma is characterized by a certain accumu-
lation of acetyl-CoA due to the fact that its entry into the tricarboxylic acid
cycle is less rapid than in other tissues. Associated with this peculiarity is
another, which is the existence of a side reaction in which two molecules of
acetyl-CoA condense together and lose a molecule of CoA to form aceto-
acetyl-CoA which is hydrolysed by a deacylase into acetoacetic acid and
CoA. This reaction only occurs to a slight degree in most cells and the
small amount of acetoacetic acid formed is reconverted into acetoacetyl-
CoA, but in the liver cell this reconversion only occurs to a slight extent.
The slowness of oxidation of acetyl-CoA, the concentration of which is
maintained at a higher level than in other cells, leads to a greater production
of acetoacetic acid which is only slightly reconverted to acetyl- CoA, and
hence ketone bodies are formed. In fact, in the liver cell, the greater part
of the acetoacetic acid formed is reduced to j8-hydroxybutyric acid by

THE INHERITANCE OF BIOCHEMICAL CHARACTERISTICS 309

butyric dehydrogenase. A small part of the acetoacetic acid decarboxylates
spontaneously to form acetone. The name ketone bodies is given to the sum
of acetoacetic acid, j8-hydroxybutyric acid and acetone. Ketogenesis,
although occurring to a slight extent in cells in general, is not accompanied
by an accumulation of ketone bodies as takes place in the liver cell. The
stationary state in normal liver is characterized by a higher concentration
of these substances which then pass into the circulation. The conglome-
ration of cells of the liver parenchyma forms the chief site of synthesis of
lipides from carbohydrate and the liver cell is responsible for the synthesis
of the phosphohpides of blood plasma.

When we come to consider protein metabolism, the cells of the hepatic
parenchyma in mammals are seen to possess important biochemical
characteristics. They are the principal site of deamination of amino acids
due to their high concentration of glutamic dehydrogenase. This process
also occurs in the kidney but the liver, which is also the site of ureogenesis,
is distinguished by possessing the system of enzymes which operate the
"ornithine cycle". In this cycle glutamic acid, ammonia and CO2 combine
to form carbamylglutamate, which in the presence of ATP and Mg++
combines with a further molecule of CO2 and a further molecule of
ammonia to give an intermediate whose nature is still unknown. This latter
compound reacts with ornithine giving citrulline and regenerating car-
bamylglutamic acid.

HOOC— CH2— CH2— CH— COOH + CO2+NH3

NH2

— H2O
glutamic acid ^HOOC— CHa— CH^— CH— COOH

NH

i
carbamylglutamic acid C — NHj

II
O

HOOC— CH2— CH2— CH— COOH

NH +NH3 + CO., — — —~> unknown intermediate

I
C— NH,

page 309, line 20 ..." an intermediate whose nature
is still unknown," should be replaced by " an inter-
mediate which is carbamyl phosphate ".

page 309, formulae .... " unknown intermediate "
should be replaced by " carbamyl phosphate ".

-j-caruaniy igiuLaiiin^ av_nj

Under the influence of an enzyme system that functions in the pre-
sence of Mg++ and ATP, the citrulline reacts with aspartic acid forming

310 UNITY AND DIVERSITY IN BIOCHEMISTRY

arginosuccinic acid which is converted into arginine and fumaric acid in the
presence of another enzyme system

H
H„N— C— N— CHa— CH2— CH2— CH— COOH HOOC— CH^— CH— COOH

II I + I

O NHa NH,

citruUine aspartic acid

enzyme H

>HN= C N— CHa— CH2— CH2— CH— COOH

Mg++,ATP 1 I I

HN— H— COOH NHa

C

I
CHa

I
COOH

arginosuccinic acid
H2O enzyme

H
H,N— C—N—CH2—CH2—CH2— CH— COOH H-HOOC—CH=CH— COOH

II i

NH NH2

arginine fumaric acid

The arginine is hydrolysed to urea and ornithine in the presence of arginase

H
H,N— C— N— CHj— CH2— CH2— CH— COOH

11 I

NH NHa

arginine

NHj

arginase /

>0=C +NH2— CH2— CHjj— CH2— CH— COOH

\ I

NH3 NH2

ornithine

The fumaric acid passes into the tricarboxyHc acid cycle or in the presence
of fumarase it is transformed to malic acid w^hich also enters the Krebs
cycle.

In mammalian liver parenchyma cells, the synthesis of the steroid
skeleton which was described on p. 236 is extended by acquisition of an
enzyme system leading to the production of the substances known as bile
acids. In man, this synthesis yields cholic acid, desoxycholic acid and
chenodesoxycholic acid. The hepatic parenchyma in mammals also con-
jugates these bile acids with taurine and with glycine.

The pathway from cholesterol to cholic acid implies that there are a
number of stages and a number of enzymes involved. Another character-
istic of the liver cell is its ability to bring about detoxication reactions, in
man the most important being conjugation with glucuronic acid. Liver
contains UDP-glucuronate and an enzyme which catalyses its formation
fronri glucuronic acid. This UDP-glucuronate appears to be one of the
substances involved in the biosynthesis of mucopolysaccharides. In the

THE INHERITANCE OF BIOCHEMICAL CHARACTERISTICS 311

transamination

aspartic acid

4-COs
+NH,

fumaric acid

glutamic acid succinic acid
+ NH.

— malic acid

presence of alcohols, phenols, or aromatic acids, the UDP-glucuronate is
diverted from its normal function and the foreign substance is excreted as
a glucuronate formed in the following type of reaction

UDP-glucuronate + ROH > UDP + R-0 -glucuronate

Bilirubin resulting from the breakdown of haemoglobin in the cells of the
reticulo-endothelial system passes into the blood plasma where it circulates
in combination with the a^-globulin fraction ; from there it passes into the
cells of the hepatic parenchyma which conjugate the bilirubin with glucuro-
nic acid and excrete the product in the bile. Furthermore the hepatic
parenchyma is the site of considerable protein synthesis, that of the blood
plasma proteins.

The cells of the parathyroid gland show a special type of biochemical
differentiation in that they are able to synthesize parathyroid hormone, a
true protein hormone which has so far not been isolated in a pure state.

The pancreatic islets consist of two types of cell differing in their bio-
synthetic characteristics : the a-cells which produce glucagon and the

page 311, diagram, the zoords "unknown intermedi-
ate " should be replaced by " carbamyl phosphate ".

312 UNITY AND DIVERSITY IN BIOCHEMISTRY

^-cells which produce insuhn. The latter hormone is a protein (see p. 107)
whilst glucagon is a polypeptide in which the amino acid sequence is the
following :

His. ser. glu. gly. thr. phe. thr, ser. asp. tyr. ser. lys. tyr. leu. asp. ser. arg.
arg. ala. glu. asp. phe. val. glu. try. leu. met. asp. thr.

(Brower, Sinn, Staub and Behrens)

The cells of the thyroid gland fix I ~ ions and oxidize them enzymically to
iodine. They contain the specific enzyme systems for the formation of
iodotyrosine and iodohistidine, and for the condensation of the iodotyro-
sines into iodothyronines, especially the two thyroid hormones, l-3:5:3'-
triiodothyronine and L-3:3'-diiodothyronine. Another thyroid hormone
and the most important quantitatively is L-thyroxine, derived from the
coupling together of two molecules of L-3:5-diiodotyrosine.

From the mesoderm are formed the cells of the muscles, of connective
tissue, the cells of the adrenal cortex, of the gonads and the urinary tract.

The cell of striated muscle is specialized chiefly for the biosynthesis of
myosin, and in the non-myosin fraction of striated muscle fibres we find
that the glycolytic enzymes predominate, hence the great importance of
aerobic glycolysis in skeletal muscle. The fibres of the myocardium are better
provided with mitochondria and the enzyme-systems for respiration and
oxidative phosphorylation than those of skeletal muscle.

The internal framework in mammals consists of connective tissue,
cartilage and bone. These structures contain and bound the groupings of
specialized epithelial cells — which are localized into organs which is turn
are integrated to form the complete organism. From a biochemical point of
view, the body framework is a network of connective tissue modified in
certain regions to form cartilage and bone.

The connective tissues are made up of three types of constituents : cells,
fibres and ground substance. In ordinary connective tissue, the cells are of
two types, histiocytes and fibrocytes. The histiocytes are provided with a
number of special biochemical properties notably in the possession of a
system able to rupture the a-methene bridge in the haemoglobin of senile
(120-130 days old) erythrocytes which they have phagocytized.

The fibrocytes are differentiated so that they can perform the biosyn-
thesis of connective fibres and the ground substance. The collagen fibres,
produced by the fibrocytes, differ markedly from the other proteins in the
mammalian organism in containing unusually large amounts of glycine and
proline, a small amount of histidine and almost undetectable amounts of
tryptophane and cystine. Also, collagen contains certain amino acids which
are not present in other body proteins (elastin excepted): these are hydroxy-
proline and hydroxylysine. Collagen fibres have a special type of physical
structure and under the electron microscope a transverse striation can be

THE INHERITANCE OF BIOCHEMICAL CHARACTERISTICS 313

seen. Elastin fibres, also a product of the fibrocytes, like collagen, contain
much glycine and proHne, traces of tyrosine and no tryptophan. But whilst
collagen is more or less "normal" in the amounts of arginine, lysine,
aspartic acid and glutamic acid which it contains, elastin contains only very
small amounts of these polar amino acids. The ground substance, also
synthesized by the fibrocytes, consists of mucoproteins the prosthetic
groups of which consist of chondroitin sulphate joined to galactosamine
by a glycoside linkage. The ground substance also contains free muco-
polysaccharides, the chief one being hyaluronic acid (acetylglucosamine
-f glucuronic acid).

In the type of connective tissue known as cartilage, the ground substance
is more dense than that of ordinary connective tissue and besides collagen
and elastin fibres it contains a chondromucoid the prosthetic group of which
is chondroitin sulphate. In the case of bony tissue, its character is a result
of the biochemical differentiation of the large cells of mesench^Tnatous
origin, the osteoblasts. The beginning of this differentiation of a mesen-
chymatous cell into an osteoblast, depends on the position of the cell in
the bone-forming region and is marked by the appearance of alkaline
phosphatase in the nucleus and the accumulation of large amounts of
glycogen in the cytoplasm. The mitochondria increase in number and the
amount of RNA in the cytoplasm also increases. Between the cells, the
interstitial substance is made up mainly of reticular fibres. As differentiation
continues, phosphatase activity appears in the cytoplasm and finally reaches
a maximum and so does the accumulation of glycogen. The number
of mitochondria continues to increase. The basophihc character of the
cytoplasm also increases. Bundles of osteogenous collagen appear in the inter-
stitial substance, distinct from normal collagen in giving a positive reaction
for alkaline phosphatase and in being highly metachromatic due to the pre-
sence of polysaccharides. The osteoblast having reached its final form the
phosphatase activity in the nucleus and cytoplasm decreases sharply and the
cytoplasmic glycogen disappears whilst the basophilia attains a maximum.
The organic matrix of the bone, strongly metachromatic and later to be
filled with bone salts, appears in the region of the non-nucleated extremity
of the osteoblast. What we have just described is the most simple type of
ossification and is known as the intramembranous type. In most bones, the
production by the osteoblasts of the calcifiable matrix of the bone takes
place on a cartilaginous former. The bone is constantly eroded and new
osseous material laid down. The erosion is due to the activity of the osteo-
clasts, mesenchymatic cells differentiated to possess proteolytic activity.
In fact, in the case of osteoblasts and osteoclasts, it would be preferable to
speak of biochemical modification rather than of biochemical differentia-
tion. In reality osteocytes, osteoblasts and osteoclasts are examples of
reversible modifications of the same type of connective cell.

314 UNITY AND DIVERSITY IN BIOCHEMISTRY

The mesenchymatic cells are not only transformed into fibrocytes,
cartilaginous cells, osteoblasts and osteoclasts. They also give rise to the
various types of blood cell. An erythrocyte begins as a reticulated cell in
the bone marrow, and, from a biochemical point of view, we can recognize
three phases in its differentiation. In the first stage, the cell contains a few
mitochondria and a nucleus with a very distinct nucleolus. The cytoplasm,
rich in RNA, is highly basophilic. The cell multiplies rapidly and an active
synthesis of proteins and nucleic acids is going on. In the second stage the
nucleus loses its nucleolus and the basophilia of the cytoplasm diminishes.
During the third stage, that of the polychromatic erythoblast, we see a fur-
ther diminution of RNA and at the same time the cell becomes acidophilic
and is coloured by acid stains. This is the result of the biosynthesis of a
new relatively basic protein, globin. At the same period, molecules of haem
and haemoglobin are rapidly formed.

In the orthochromatic erythroblast, or normoblast, cell division does not
occur. The mitochondria have disappeared and only traces of RNA remain in
the c\i:oplasm. The nucleus is small. On the other hand, the haemoglobin con-
centration has increased from 1-2 parts per 100 to 20 parts per 100. The
nucleus having disappeared, the cell flattens and becomes biconcave so that
the red blood cell is nothing more than a bag of haemoglobin, only the cyto-
plasmic enzyme systems having been retained : those for glycolysis and the
hexosemonophosphate shunt, reducing systems able to maintain the
haemoglobin in the reduced state. The red blood corpuscle contains
relatively large amounts of catalase. It protects the haemoglobin by
decomposing the hydrogen peroxide which would otherwise form in the
corpuscle.

The study of leucocytes is still in the hands of histologists. But a few
results of a biochemical nature have been obtained and they show some
aspects of differentiation. Neutrophil leucocytes, for example, show a high
aerobic glycolysis and they contain esters of hyaluronic acid. The granules
of the eosinophils are surrounded by an envelope of phospholipides and
among their constituents there is an antihistaminic substance. From the
megakaryocytes of the bone marrow are derived the blood platelets which
are highly specialized biochemically. They contain, and liberate into the
blood on clotting, a number of platelet factors, one activating thrombo-
plastin, another accelerating the formation of thrombin from prothrombin,
yet another accelerating the conversion of fibrinogen to fibrin, etc.

The most remarkable example of differentiation in cells of mesodermic
origin is the metabolic series which continues the process of biosynthesis
of the steroid ring (see p. 236). We have already described one of these
continuations, the synthesis of bile acids by the liver cell. In the cells of
the adrenal cortex and the gonads, other systems continue the biosynthesis
of the steroid ring with the formation of steriod hormones.

THE INHERITANCE OF BIOCHEMICAL CHARACTERISTICS 315

It is generally accepted that the active cells in the adrenal cortex are those
of the zona fasciculata, which secrete corticosteroids under the influence of
ACTH. The secretion of the various corticosteroid hormones implies that
a complex system of enzymes is involved which characterizes the biochemi-
cal differentiation of the cells of the zona fasciculata. The reader will find
an excellent account of the biology of the adrenal cortex in the recent work
of Chester Jones (1957).

When we come to consider the biosynthesis of the steroid hormones by
the gonads, or by the placenta, here also we have enzyme systems special
to these cells.

The preceding pages give only a slight idea of the biochemical diversity
accompanying cellular differentiation in the organism of a mammal such as
man. Also, the description of these differentiations has been made in an
altogether too schematic manner without taking into account the variations
existing from cell to cell in the framework of the same cellular
differentiation.

Among the same class of differentiated cells we may also observe an
individual biochemical variation depending on time, age and the reproduc-
tive cycle, etc. In the water flea Daphnia, for example, the haemoglobin
content of the blood varies with the instar. The smallest concentration
corresponds to the time when the eggs have reached the development of
late-stage embr}'os ready to be released. After this the blood haemoglobin
rapidly diminishes, passing from the blood into the ovaries before the
eggs are laid. After the laying of the eggs the blood of the female
gradually recovers its haemoglobin content (Fox, Hardcastle and Dresel,
1949).

III. PHENOTYPE AND ''MILIEU"

Under given conditions of the milieu, cellular differentiation controlled
by a given genotype can produce an extreme diversity of results in the way
of biochemical systems. When to this is added the influence of the milieu
even further differences in a given biochemical system may be produced.

Grasshoppers which have been raised at an elevated temperature are
pale whilst those which have grown at a lower temperature are dark. This is
because at temperatures higher than 40° the biosyntheses of m.elanin and of
insectorubin are inhibited. Here we have a direct action on an enzymatic
component of the existing phenot}^pe, and we may call this a phenotropic
action of the milieu.

A further example, the green pigment present in the tegument of solitary
locusts is not present in the insects of the gregarious type. The haemolymph
of the solitar}^ form is a brilliant green whilst that of the gregarious form is
a golden-yellow. The characteristic difference here is the production of
mesobiliverdin in the solitar\' locust.

316 UNITY AND DIVERSITY IN BIOCHEMISTRY

A remarkable example of the influence of the milieu on the biochemical form
of the phenotype is provided by the influence of the partial pressure of oxy-
gen in the milieu on the biosynthesis of haem derivatives. It has been shown
in yeasts that the presence of air stimulates the synthesis of cytochromes a, b
and c and that the absence of air inhibits this synthesis. On the other hand,
it is vjtW known that a fall in oxygen pressure increases the blood haemo-
globin in man at high altitudes and in fishes living in waters poor in oxygen.
In mammals, a fall in the external oxygen pressure also increases the cyto-
chrome-c and myoglobin concentrations in muscles. Among the Cladocera,
a subdivision of the Crustacea, a fall in the oxygen content of the medium
increases the haemoglobin concentration in muscles, ganglia and blood. In
Daphniapulex, D. magna and D. obtusa, an increase in blood haemoglobin in
poorly aerated water has been demonstrated by H. M. Fox. Fox and Phear
also observed that the animals lost their blood pigment in well aerated waters.

Whatever the type of differences in the biochemical order which can be
brought about by the influence of the medium on the biochemical phenotype,
it is possible to determine the spectrum of possible phenotypes and so to find
the limits set to the organism by its own genetic potential in the realization
of the phenotype by the interaction of the environment and the genotype.

REFERENCES

Baker, J. R. (1948). The cell-theory : a restatement, history and critique. Part I.

Quart, y. Micr. Sc, 89, 103-125.
Jones, T. Chester (1957). The Adrenal Cortex. Univ. Press, Cambridge.
Knox, W. E., Auerbach, V. H. and Lin, E. C. C. (1956). Enzymatic and metabolic

adaptations in animals, Physiol. Rev. 36, 164-254.
Prosser, C. L. (1955). Physiological variation in animals. Biol. Rev., 30, 229-262.
Wagner, R. P. and Mitchell, H. K. (1955). Genetics and Metabolism. Wiley,

New York.

CHAPTER III

BIOCHEMISTRY AND TAXONOMY

I. BIOCHEMICAL DIVERSITY

"Each species consists of groups of individuals with more or less similar
gene combinations, optimally adapted for a given environment" (Mayr,
1949). This definition of species implies the existence of biochemical
characters typical of the species and that these specific characters are
adapted to the ecological niche in which the species prospers and dominates
its competitors.

To illustrate this, let us consider the special properties of the haemo-
globin in animals.

Typically, the respiratory function of the internal environment operates
by means of a cycle in which the internal environment circulates around the
organism between the various tissues and the point where it is equilibrated
with the external environment or a continuation of the latter.

At the point of contact with the external environment the blood becomes
charged with oxygen and gives up carbon dioxide. On reaching the tissues
it loses oxygen and takes up carbon dioxide. This typical respiratory cycle
requires the mediation of a physico-chemical system to bring about the
transport of oxygen in one direction and of carbon dioxide in the other.

There are a number of properties of haemoglobin which are important
when it functions as a carrier. It can be oxygenated and the degree of
oxygenation is a function of the partial pressure of oxygen. Being a hetero-
protein, it bears acid groups which can combine with bases, the dissociation
of these groups varies with pH and cations are lost when the pH approaches
the isoelectric point. The dissociation does not only vary with the pH but
also according to the degree of oxygenation, and inversely, the degree of
ox}^'genation at a given partial pressure of oxygen will vary with the disso-
ciation of the acid groups in the neighbourhood of the oxygen-bearing
groups. On the other hand, the carrier molecule bears free — NHg groups
which are able to form compounds of the carbamate type with carbon
dioxide.

We may plot the amount of absorbed oxygen as a function of its partial
pressure for different bloods and coelomic fluids, the temperatures and
carbon dioxide partial pressures being those existing in the arterial blood.

317

318

UNITY AND DIVERSITY IN BIOCHEMISTRY

If we do this we obtain the series of curves shown in Fig. 89. These curves
are very different one from another, one of the reasons for this being that
the number of oxygen-binding groups varies from case to case. The oxygen
capacity, i.e. the quantity of oxygen in volumes per cent corresponding to
complete saturation of the carrier, in effect regulates the level at which the
curve flattens out. In order to compare the different curves with profit, it is
necessary to consider in each case, for different partial pressures, not only
the amount of oxygen combined with the carrier, but also the degree of
saturation of the latter. Thus, in Fig. 90 we have a series of curves whose
positions can be related to each other by noting the value of the partial
pressure of oxygen corresponding to 50% saturation (p^o)- The position

Table XIX

Rata
ocellata

Chelydra
serpentina

Duck

Goose

Man

Oj total transported, vol. %
O2 transported in dissolved
form, vol. %

3-92
0-22

3-70
0-17

100
0-15

5-14
0-17

5-30
010

P02 = ± 150 (in air, or in water in equilibrium with air)

Table XX

S

Q

<n

■^ a

^

G «

K

•>.

Q

s

f^

Q
K

S

<3

le-

to

Q

a

•>-»

Q

^

^

<j

s

-^

,2

VJ

* *>4

0

0

^

K
S

s

0

.a

3

CO

0
0

c

0

CC

b

cq

^

ct;

0

Q

0

^

X

Hr

Hb

Hey

Hey

Hb

Hb

Hb

Hb

Hb

Hb

/>Os arterial

32

75

36

115

70

57

102

94

100

78

% sat. arterial blood

90

97

95

97

93

95

98

96

98

96

and the shape of the curves in Fig. 89 correspond, in each case, to the
temperature and CO2 partial pressure of the arterial blood. In the case of
the ray the temperature is 10° and in man 38°.

The partial pressure of CO2 is 8mm in the frog and 43mm in the case
of the Urodel Amphiuma.

If the curves had been traced at the same^^Q^, and at the same tempera-
ture, the ^60 values would not have been those shown in Fig. 90. The
particular shape and form which correspond to physiological conditions is

BIOCHEMISTRY AND TAXONOMY

319

Fig. 89 — Oxygen absorption curves for various bloods and coelomic fluids con-
taining haemoglobin under "arterial" conditions reproduced in vitro. The
black circles indicate the value of the arterial poi-

100

10 20 30 40 50 60 70 80 90 100

Fig. 90 — Oxygen absorption curves for a number of "milieux interieurs" con-
taining haemoglobin. The temperature is given and the pH except in the case
of the Frog and Amphiuma, where the partial pressure of carbon dioxide

is listed.

320 UNITY AND DIVERSITY IN BIOCHEMISTRY

the result of the action of a muhitude of factors such as the p^^Q and tem-
perature of the arterial blood, the affinity of the haemoglobin for oxygen,
the intensity of the Bohr effect, etc.

Corpuscles containing haemoglobin are found in the blood of all Verte-
brates, and since these animals have a well-defined respiratory cycle they are
a useful starting-point for the study of the role of haemoglobin in the
transport of oxygen by the blood.

The important part played by haemoglobin in oxygen transport is clearly
shown by the values obtained for different bloods of the "transported
oxygen", i.e. the difference in total oxygen between the venous and arterial
blood less the difference in dissolved oxygen for these bloods.

The part played in oxygen transport by the sigmoid character of the
dissociation curve is evident. With the partial pressures of oxygen occurring
in arterial and venous blood, a hyperbolic curve would only allow the
transport of a much smaller amount of oxygen. When the reduction of the
blood is accompanied by acidification the Bohr effect also comes in, since
the curve is displaced to the right, so that, at the same pQ^ there is a lower
degree of oxygenation of haemoglobin.

Does the dissociation curve, as it exists under arterial and under venous
conditions, show any characteristics parallel to the physiological or ecolo-
gical character of the animal?

If, regardless of the particular oxygen carrier they contain, we examine
the few animal species whose blood respiratory cycle is known, and if we
note the values of the arterial p^^ and the degree of saturation of the arterial
blood, we shall find that from one case to another there exists, even for
animals living in the same environment, very different gradients of oxygen
pressures, contrary to what is found with CO2 which is always, or nearly
always, in the arterial blood, in equilibrium, with the external environment.
Yet, whatever the p^^ resulting from the particular character of the res-
piratory system, the arterial blood is always found to be 90 to 98% saturated.
In other words, whatever the difference between the external Pq^ and the
/>o^, the point on the dissociation curve corresponding to the arterial Pq^
is always found to lie along the top right-hand portion of the curve.

Hence the shape and position of the dissociation curve are adapted to
the respiratory needs of the organism. The slightest decrease in the />o,
would immediately bring about a loss of oxygen from the haemoglobin.
The fact that one is not justified in considering, even in aquatic animals,
that the arterial /)q, is equal to the external ^q_ has been verified by the
observations of H. M. Fox (see Table XVII).

Despite the fact that we know little about the respiratory cycle of fish,
it is possible to establish a relation between the more or less vertical shape
of their dissociation curve and the concentration of oxygen in the external
environment. First proposed in 1919 by Krogh and Leitch, this relation has

BIOCHEMISTRY AND TAXONOMY 321

been confirmed many times. As these authors pointed out, a strong affinity
for oxygen allows a fish hving in badly aerated water to charge its blood
with oxygen more easily than could a fish possessing a haemoglobin of low
affinity. Moreover, a high affinity, accentuated by a low temperature, would
maintain the curve in the left-hand region between the axes, i.e. in the low-
pressure region, and consequently the oxygen would be released to the
tissues at a low pressure, unless the Bohr effect was to interfere and displace
the curve to the right. Several workers have confirmed that in fresh-water
fish the Bohr effect is more marked than in those living in deep cold waters
and that the blood of these same fish also has a lower affinity for oxygen at
low />CQ^ values. These two characteristics, feeble affinity and a relatively
powerful Bohr effect, counterbalance the properties imposed on the blood
by the low temperature. Various observations reveal the more or less
marked "pumping action" due to the Bohr effect, in deep-water fish, in
bringing about the liberation of oxygen in the swim-bladder. But if the
Bohr effect is more marked in fresh-water Teleosts than in mammals, it is
still more pronounced in the marine Teleosts which fits in with a character-
istic property of the marine Teleosts, that of having a low arterial ^co,»
corresponding to that of sea water. Here, the Bohr effect appears to be an
effective correction of the almost hyperbolic character of the dissociation
curve, without decreasing the efficiency of the charge mechanism as would
be the case in a medium rich in COg.

Another example of a relation between the affinity for oxygen of the
haemoglobin in the arterial blood and the oxygen level of the environment is
provided by mammals living at high altitudes, such as the lama {Lama
huanachus), the vicuna [Lama vicugfia) and the viscacha [Lagostoma sp.),
the haemoglobins of which have a higher affinity for oxygen than those of
other mammals.

When the venous blood leaves the tissues on its way to the heart, the
partial pressure of oxygen is 40mm Hg for man, 38mm in the horse, 56mm
in the goose, 37mm in the duck, 15mm in the turtle Chelydra serpentina,
14mm in the ray Raia ocellata, and the corresponding degrees of saturation
are 60%, 70%, 38%, 35% and 33% respectively. Thus, on the one hand a
sufficient ox}^gen concentration gradient is assured, this being an important
factor in the supply of this element to the cells, and on the other hand, the
venous blood carries a reserve of oxygen which can be called upon if the
rate of metabolism is increased, or if the supply of oxygen is curtailed.

The part played by this reserve is prominent in the seal which, during a
dive, uses almost the whole of the oxygen in its blood. The almost total
reduction of the venous blood has been reported to occur in other diving
animals such as the duck and the musk-rat.

The use of haemoglobin to provide a reserve of oxygen can be observed
duringperiodsof suspension of the breathing mechanism. In an Invertebrate

w

322 UNITY AND DIVERSITY IN BIOCHEMISTRY

not provided with a circulation like Urechis, the functioning of this
reserve can be clearly seen. The Echurian Urechis caupo is found along the
Californian coast living in the mud and sand in a U-shaped tube through
which it causes water to circulate during its periods of activity by the
peristaltic action of its musculo-cutaneous tube. The continually renewed
water brings food and oxygen to it. In fact by a peristaltic movement oppo-
site in direction to that of the musculo-cutaneous tube, the animal can
"inspire" the circulating water through its anus into its terminal intestine
which is highly developed and the thin wall of which is in contact with the
haemoglobin-containing corpuscles of the coelomic fluid. After a number
of these inspiratory movements, a single expiratory movement follows
expelling all the water in the intestine. In well-oxygenated water the haemo-
globin of the coelomic fluid is almost completely saturated (97%) although
the oxygen partial pressure is only 75mm Hg i.e. much less than in the
surrounding water (around 150mm). If the animal is at rest under these
conditions its haemoglobin will remain completely saturated and the
dissolved oxygen will be sufficient for its needs. As we have said, the animal
draws sea-water into its intestine and then expels it after having oxygen-
ated its coelomic fluid at the expense of the oxygen in the sea- water. (In the
expired water pQ_^ — 100mm approx.) Under these conditions it does not
use its haemoglobin at all. The volume of the coelomic fluid is about 20cm^
and its oxygen capacity is 4 volumes per cent, so that the coelomic fluid
contains 0*8 cm^ of oxygen. The oxygen consumption of Urechis, on an
average, is around O-OlcmYmin. It is clear that only a small part of the
oxygen has to be replaced each minute and for this the dissolved oxygen
suffices. During its periods of activity which coincide with the periods of
elimination of water, Urechis lives on the dissolved oxygen provided by its
respiratory system. When the animal retires into the middle horizontal part
of its tube after a period of activity, it rem.ains motionless and ceases to
cause the water to circulate or to be taken into its digestive tube.

The total amount of oxygen in the coelomic fluid and in the water of the
intestine corresponds to the amount which it consumed in 70 min; if the
coelomic fluid did not contain haemoglobin the oxygen would be used up
after 14 min. Thus the presence of haemoglobin enables the animal to have
a rest period between its periods of activity five times longer than would
otherwise be possible.

When Urechis respires, the pQ^ of the water in its intestine is lOOmm
when the /)q^ of the outside water is 150mm.

Under these conditions the pQ^ of the coelomic fluid is 75mm. These
conditions correspond to 97% saturation. The haemoglobin in the cor-
puscles remains saturated and the oxygen diffuses from the water in the
intestine to the blood plasma and from there to the tissues. There is no
evidence of the haemoglobin, which does not circulate, playing any part as

BIOCHEMISTRY AND TAXONOMY 323

a carrier. During the periods of rest, the animal stops breathing. The
ox}^gen is not replenished from the water in the intestine and consequently
the Pq_^ of the coelomic plasma tends to fall, thus bringing about a liberation
of oxygen from the haemoglobin because of the particular position of the
dissociation curve.

II. DIVERSITY WITHIN SPECIES
To define a species as consisting of groups of individuals with more or
less similar gene combinations m.eans that there are genotype differences
within the species itself. In the human species, which has a high variability,
we may say that there are no two individuals, even twins, possessing the
same assortment of genes. A large amount of data is available on individual
variation in humans (see R. J. Williams, 1956). The water concentration in
18 normal men and 11 normal women was found to vary from 45*6% to
70*2%. We also have a number of results on the quantitative pattern of
various enzyme systems. The plasma alkaline phosphatase concentration,
is a characteristic of the individual and remains constant, but from one
individual to another it was found to vary between 1*29 and 14-0 units
(of 600 subjects examined by Clark and Beck). The cholinesterase of red
blood corpuscles is constant for a given individual but varies considerably
from one person to another, etc.

III. DIVERSITY BETWEEN SPECIES
It is to Aristotle that we owe the invention of the first practical system
for the classification of living beings. More than 2000 years ago this natura-
list of genius proposed to classify organisms according to the degree of
similarity of their morphological and anatomical characteristics. Despite
the appearance of other systems claimed to be more natural, but today
forgotten, the system of Aristotle has survived and is referred to today as
"the natural system". A certain number of species are collected together
into a new group, defined by the possession of common characteristics,
and several of these groups together form a more general category, etc.
By the accumulation of taxonomic data it was possible to formulate a
principle which was the subject of much admiration and satisfaction among
naturalists; this was, that classification, based upon a few diagnostic
characteristics, of a species and the placing of it in a given class enables one
to forecast the existence of a whole range of morphological and biological
traits in individuals of that same species. This idea, to which we are
accustomed today and which appears self-evident and banal, must have
stirred those few who glimpsed the possibilities which at that time were still
unsuspected by the majority. When finally the merit of this system was
admitted, it was recognized that an essential criterion was the existence of
a fundamental similarity and not merely a superficial one it also led to the
recognition of a common basic plan in the chief natural groups. During

324 UNITY AND DIVERSITY IN BIOCHEMISTRY

the first half of the 19th century, this idea appeared in numerous guises
depending on the personal preferences of its protagonists. For the most
theoretically inclined, like Goethe or Oken "these ideal patterns which the
creative principle set before itself were, so to say, Platonic ideas in the mind
of the creative spirit" (Sir Charles Sherrington. Goethe on Nature and on
Science. Cambridge, 1949, p. 24). The more objective, as Julian Huxley
said of Thomas Huxley, "simply assumed that structural homology (or
common archetypal plan) was the right key to unlock classificatory secrets"
(Julian Huxley. Evolution. The modern synthesis, London, 1942, p. 391). We
might as well say that during the first half of the 19th century, the idea
of a plan was purely descriptive and had no explanatory basis, and that
Schiller was right when he repHed to the exposition of Goethe : "Das ist
keine Erfahrung; das ist eine Idee."

The publication of the great work by Charles Darwin changed all this.
Since the advent and generalization of the Evolution Theory, the basic
criterion for natural classification, which up till then was the degree of simi-
larity, has become the degree of phylogenetic kinship. The aim of present
day taxonomy is to build up a classification consisting of classes based upon
phylogenetic relations. As Dobhzansky has pointed out, this new point of view
has not led to any fundamental modification of classification. Here is an
important point, the consideration of which leads to an increased confidence
in the system established by taxonomists, which we shall use as our guide.

When discussing the significance of various characteristics of plants useful
in their taxonomy at the supraspecific level, Erdtmann (1952) wrote: "Much
attention is paid by taxonomists to minor characteristics such as the form,
structure and arrangement of epidermal cells because they are essentially
indifferent to external factors and consequently conservative. Such proper-
ties are therefore handed down from generation to generation and from
species without suffering much change. Comparatively recent specializa-
tions possess little taxonomic interest."

From the biochemical aspect, as with other aspects, if it is true that the
characteristics of the species and its sub-divisions are naturally inserted into
the physiology of the organism and its relation with its ecological niche, it
is to be expected that the biochemical traits which are transmitted from
organism to organism and bear witness to a common line of ancestors will
not be those of an essential physiological or ecological character. The
utility of supraspecific categories is above all to enable us to express our
views on the probable nature of phylogeny. As Caiman (1949) says, "the
characters most important in taxonomy are those which maintain them-
selves unchanged through the greatest range of variation."

The higher categories will thus indicate those limits within which
characters common to a more or less broad range of species maintain them-
selves in spite of great species, physiological and ecological differences. The

BIOCHEMISTRY AND TAXONOMY

Table XXI

The principal pigments of the different classes of algae

(compiled by G. E. Fogg, 1952)

325

y
>.

a
2

u

—

c
o

Si

a

a

C

'2

W

a
o

V

a
Oh

Bacilliariophyceae

u

C

0

c

ca
w
u

o
s:

c

«

X

Dinophyceae

Rhodophyceae

Myxophyceae

Chlorophylls

Chlorophyll a

«

•

•

•

9 (

»

•

• <

9 4

i

Chlorophyll b

3

«

o

-

-

-

-

-

-

-

Chlorophyll c

-

-

-

O

O

-

o

-

-

Chlorophyll d

-

-

-

-

-

-

-

D

-

Chlorophyll e

—

—

—

—

—

o

—

-

-

Carotenes

a-carotene

o

•

-

-

•

- (

D

•

jS-carotene

•

o

•

9

9 <

»

9

• <

9 i

»

E-carotene

—

o

•

—

O

•

•

Xanthophylls

Lutein

•

o

?

-

- (

D

-

- (

)

?

Zeaxanthin

o

•

-

-

-

-

»

P

Violaxanthin

o

o

O

-

•

-

Flavoxanthin

?

?

O

-

•

•

Neoxanthin

o

a

o

-

-

-

Siphonein

-

o

-

-

-

-

-

Siphonoxanthin

•

o

-

-

-

-

-

Fucoxanthin

-

-

—

3

3 (

D

-

-

Neifucoxanthin

-

-

-

O

O

-

-

Diatoxanthin

-

-

-

?

O

-

-

Diadinoxanthin

-

-

-

?

O

-

o

Dinoxanthin

-

-

—

?

—

-

o

Neodinoxanthin

-

-

-

-

-

-

o

Peridinin

-

-

-

-

-

-

3

Myxoxanthin

-

-

-

-

-

-

-

C

>

Myxoxanthophyll

-

-

-

-

-

-

-

(

>

Unnamed

•

•

o

o

[

3

•

' i

>

Phycobilins

r-phycoerythrin

-

-

-

-

- r

-

«

>

r-phycocyanin

-

-

-

-

- ?

-

c

:>

c-phycoerythrin

-

-

-

-

- r

-

-

c

5

c-phycocyanin

"

"

"

"

" '

"

~ "

f

»

326

UNITY AND DIVERSITY IN BIOCHEMISTRY

phylogenetic implications of this concept, or at least, to be more cautious,
its importance with respect to classification, is obvious.

Even as early as 1854 the Austrian chemist Rochleder clearly stated the
taxonomic importance of the biochemical characteristics of plants, where
the more general classes were concerned, thus : "Die Familienahnlichkeit

Table XXII

Biochemical characteristics of the algal classes

(compiled by G. E. Fogg, 1952)

Class

Reserve

Cell wall

Sterols

products

constituents

Chlorophyceae

starch

cellulose

sitosterol

fat

pectin

fucosterol

chondrillasterol

ergosterol

Xanthophyceae

fat

pectin
silica
cellulose
chitin (?)

sitosterol

Chrysophyceae

leucosin

pectin

fucosterol

fat

silica

unidentified sterols

Bacillariophyceae

leucosin (?)

pectin

unidentified sterols

Cryptophyceae

starch

cellulose (?)

Dinophyceae

starch

cellulose

fat

pectin

Euglenineae

paramylum

none

Phaeophyceae

mannitol

algin

fucosterol

laminarin

fucoidin
cellulose

Rhodophyceae

floridoside

polygalactose-

fucosterol

mannoglycerate

sulphate esters

sitosterol

floridean starch

cellulose

unidentified sterols

Myxophyceae

myxophycean

pectin

none

starch

cellulose

cyanophycin

Higher plants

starch

cellulose

sitosterol

fat

pectin

chondrillasterol

lignin

stigmasterol

der Pflanzen is bedingt durch das gleichzeitige Vorhandensein mehrerer
Stoffreihen". Much information on the relations between the biochemistry
and the taxonomy of plants will be found in Molisch's Pflanzenchemie und
Pflanzenverwandschaft and also in the Handbuch der Pflanzetia?ialyse. Some
very interesting studies have been carried out by Erdtmann on the chemical
taxonomy of the heartwood constituents of conifers.

BIOCHEMISTRY AND TAXONOMY 327

The biochemical characteristics of various classes of algae are listed in
Tables XXI and XXII. We can see that the biochemical diversity of these
classes is clearly indicated. Moreover certain of these classes show biochemi-
cal similarities confirming the grouping of the Xanthophyceae, Chr}^sophy-
ceae and Bacillariophyceae under the heading Chrysophyta while the
affinities between the Dinophyceae and the Crj'ptophyceae justify their
grouping in the Pyrrophyta.

When we come to the higher groups in animal taxonomy, the idea of
biochemical character is by no means a new one. For a long time, the taxo-
nomists have considered the siliceous skeleton of Radiolaria as a character-
istic of that order, and within this order the presence of strontium sulphate
in the skeleton as characteristic of the sub-order Acantharia. In the phylum
Porifera, the class Calcispongiae have long been distinguished from other
sponges by the calcareous nature of their spicules. The phylum Brachio-
poda has been divided into the Ecardines, characterized by a calcareous
shell, and the Testicardines. Similarly, the presence of chitin in the tegu-
ment of Arthropods has been considered as one of the characteristics of
that phylum, and the possession of a calcareous shell as characteristic of the
Mollusca.

Several biochemical characteristics of the sub-phylum Vertebrate can
be enumerated :

(a) the biochemical system of bone, involving a number of functional
subtances such as parathyroid hormone, vitamin D, etc. ;

(b) the biochemical system of blood clotting, involving a protein
characteristic of Vertebrates, fibrinogen, and a mechanism trans-
forming fibrinogen into fibrin by the action of thrombin, resulting
from the action on prothrombin of a number of substances present
in plasma and blood platelets;

(c) the presence of keratin in the skin;

(d) the system of digestive enzymes in the form of a series of hydrolases,
starting with pepsin and acting at diflFerent points ;

(e) the presence in the blood of red cells containing a typical haemo-
globin, characterized by its molecular weight and by certain definite
proportions of arginine, cystine, histidine and lysine;

(/) the presence of a liver secreting a bile containing special steroids,

the bile salts;
(g) the protein system of blood plasma ;
[h) the presence of a carbohydrate metabolism involving insulin and

systems sensitive to its action;
(/) the presence in the blood plasma of a system of inorganic bases

and especially of a high level of sodium;

328 UNITY AND DIVERSITY IN BIOCHEMISTRY

Within the sub-phylum Vertebrates each class has its own biochemical
peculiarities, some of which are of diagnostic value. The class Cyclostoma
is so different from other Vertebrates in some of its biochemical characters
that it deserves special mention. The biochemical mechanism for ossifica-
tion is completely lacking from the Cyclostomes, and they show no sign of
calcification. Another character distinguishing them from Vertebrates in
general is the nature of the blood haemoglobin. Like the myoglobin of
Vertebrates, the haemoglobin of the lamprey has a molecular weight of
17,000, its arginine and lysine content is similar to that of vertebrate
haemoglobins whereas the histidine and cystine content as about the same
as that of an invertebrate haemoglobin. The dissociation curve of lamprey
haemoglobin is hyperbolic. The blood serum of Cyclostomes on electro-
phoresis gives a pattern which is very different from that of the other
Vertebrates. But, in spite of the above differences, the Cyclostomes have a
number of the biochemical characters of Vertebrates : a keratinized
epidermis, a liver secreting a characteristic bile and specialized for the
manufacture of glycogen, and their phosphagen is creatine phosphate as is
general for Vertebrates.

Among Vertebrates, the class of fishes is distinguished by the complexity
of their depot fats, the fatty acids in them covering a wide range of homo-
logues in 14, 16, 18, 20 and 22 C atoms. The character of the depot fat
is not only influenced by the character of the ingested fat, but also by the
ability of the particular fish to hydrogenate or dehydrogenate the fatty acids
presented to it.

In the fishes, as among most aquatic forms, xanthophylls are accumu-
lated in preference to carotenes. But among the accumulators of
xanthophylls, fishes form a special group since they accumulate only three :
lutein, taraxanthin, and astaxanthin. These carotenoids are accumulated
particularly in the chromatophores of the skin where they play a part in
photo-response.

The role of the thryoid secretion in fishes is still unknown, but it can be
said that, contrary to the case in the higher Vertebrates, the thyroid secre-
tion of fishes has no action on metabolism and, contrary to its action in the
Amphibia, it does not appear to influence metamorphosis.

Among the fishes, the sub-class Elasmobranchii, to which skates and rays
belong, possesses certain special biochemical characters. The skeleton is
essentially cartilaginous. It is calcified in some regions but properly speak-
ing it is never bone tissue which is found only in the scales and teeth. Like
all Vertebrates, the Elasmobranchii possess a liver secreting a bile which
contains steroid substances. This bile contains a special bile salt, scymnol
sulphate, a C27 steroid which has been detected in all Elasmobranchii
studied so far, while in the true fishes cholic acid, a C24 steroid, is present
in combination with taurine.

BIOCHEMISTRY AND TAXONOMY 329

The organs and blood of the Elasmobranchii contain considerable
quantities of urea. In the blood of marine Elasmobranchii, urea may attain
a concentration of 26g/l and is never less than 18g/I. Here we are dealing
with a case of selective retention of urea since the other nitrogenous com-
pounds in Elasmobranch blood are no more concentrated than in the blood
of other Vertebrates.

As in the case of the Cyclostomes, the biochemical characters of the
Elasmobranchii are so clearly different from those of the teleost fishes that
they are strong arguments in the support of those zoologists who favour
the separation of the class of Elasmobranchii from the class of fishes.

Like the Elasmobranchii and unlike teleost fishes, Amphibia have bile
salts of the C27 type. While the bile salts of teleost fishes are present in
combination with taurine, those of Amphibia are always combined as
sulphates as in the Elasmobranchii. Thyroxine has no action of the meta-
bolism of Amphibia, as in the fishes, but the thyroid secretion has a very
marked effect on their metamorphosis.

The Amphibia are uniform in their amino-nitrogen and purine cata-
bolism which is always ureotelic.

Biochemical support for the separation of the Amphibia into the orders
Anura and Urodela is found on comparison of the retinal pigments in the
two. While the retina of Urodela contains mostly porphyropsin (p. 291)
which is also present in Cyclostomes and is a derivative of vitamin Ag, the
retina of the adult Anura contain only rhodopsin, a derivative of vitamin
Aj. In this respect the Urodela are nearer to the Dipneusti which have
porphyropsin as their retinal pigment. It can be pointed out here, that in
the nature of their retinal pigment the class of fishes is very heterogeneous,
as is the case for nitrogen metabolism and a number of other characters.

The nitrogen metabolism of the class Reptilia differs from that of fishes
and Amphibia. They always have a uricotelic amino-nitrogen metabolism,
a fact connected, as stated in dementi's law, with the lack of arginase in
the liver. Another interesting point is the fact that in terrestrial turtles,
such as Testudo graeca, a more or less active system of ureotelic metabolism
is present together with the enzyme system for uricotelic metabolism. With
regard to their biliar)' steroids, the Reptilia show a systematic variation :
among the sub-class Chelonia, the biliary steroids are derivatives of
sterocholanic acid, while most of the members of the order Serpentes con-
tain chloic acid. Among these, however, the family Boidea is characterized
by pythocholic acid. Turtles and snakes contain C24 biliary steroids,
while Haslewood has found in the sub -class CrocodiHni that the alligator
has a C27 steroid.

In all Reptilia examined so far, the biliary steroids are combined with
taurine, as is the case with the teleost fishes, while in the Elasmobranchii
and Amphibia the steroids are always in the form of sulphates. Reptilia,

330 UNITY AND DIVERSITY IN BIOCHEMISTRY

like all Vertebrates, have keratin in their integument, but in addition
another keratin is found in their skin which is called "feather keratin"
because it is also found in bird's feathers. This is not the only biochemical
character that reptiles and birds have in common; both have a uricotelic
nitrogen catabolism.

Although it is true that Reptilia and birds have many biochemical
characters in common, there are also some peculiar to birds alone. Among
them is the mechanism for the detoxification of benzoic acid by synthesis
of ornithuric acid, a conjugate of benzoic acid and ornithine.

The biliary steroids of more than sixty species of mammal have been
studied. In general, the characteristic biliary steroid is cholic acid. It is
particularly interesting to find in groups of related species certain unique
bile salts, although not necessarily as the principal constituents. For
instance, in all members of the families Otaridae, Odobenidae and Phocidae
(the seals and walruses) so far examined, a special bile acid, ^-phocaecholic
acid, has been found in addition to cholic acid. Certain genera too have bile
salts of their own, for example the genus Sus which has no cholic acid but
hyodesoxycholic acid instead. As already stated, biliary steroids are con-
jugated in the form of sulphates in the Elasmobranchii and Amphibia, and
with taurine in the teleost fishes and reptiles. In mammals they are con-
jugated with taurine and glycine in varying proportions. The presence of
glyco-acids is characteristic of mammalian biles.

The members of the sub-phylum Urochorda do not show the bio-
chemical features we have described as being characteristic of Vertebrates
but they have features of their own. They possess a cellulose coat and they
accumulate vanadium in their tissues and blood cells. Although there is no
thyroid secretion, they are sensitive to thyroxine which activates the
development of the larvae. Also, their neural glands contain principles
similar to the hypertensive, melanophore-expanding, oxytocic and gona-
dotrophic principles of the vertebrate hypophysis.

All the facts so far collected pertain to the Chordates, but many facts of
a similar nature could be gathered through the study of each of the groups
of the Metazoa. In what follows we shall limit our enquiry to just a few
examples.

One of the oldest identified features of the phylum Arthropoda is the
presence in their integument of a polyacetyglucosamine called chitin. In
the integument the chitin is combined with protein. Another general
character of Arthropods is the peculiar nature of their blood coagulation
mechanism. The physiological phenomenon of blood clotting is found in
Vertebrates and Arthropods and the two systems show certain similarities.
The blood clotting mechanism in Vertebrates is summarized in Fig. 90a.

In Arthropods, the phenomenon of plasma coagulation depends upon
the action of tissue coagulins on a coagulable protein. In the lobster, which

BIOCHEMISTRY AND TAXONOMY 331

Thromboplastin + prothrombin — lateie^acclierator"*' thrombin

Plasma Ac-globulin ''''°'"^'" — > serum Ac-globulin
Thromboplastin + prothrombin ^"""^ — - — >• thrombin

platelet accelerator
serum Ac-globulin

Fibrinogen _HE£E1^_ fibri

rm

Fig. 90a (Ware, Fahey and Seegers). Events in clotting of vertebrate blood plasnr<a

is the only case studied to date, this protein is quite different from the
fibrinogen of Vertebrates. It is more soluble in salt solutions and moves
faster in an electric field. An extract of Arthropod tissue coagulates an
electrophorectically homogeneous preparation of lobster fibrinogen in the
presence of calcium. During coagulation in Arthropods under physiological
conditions, coagulin does not arise, as has long been believed, by an aggluti-
nation and disintegration of all blood cells, but from a special type of cell,
the coagulocyte, Coagulocytes are homologues of the explosive cells
described by Hardy (1892) in Crustacea.

The exocuticle of Arthropods is the result of the hardening of part of
the endocuticle containing chitin in association with a water soluble protein
to w^hich Fraenkel and Rudall gave the name arthropodin. This hardening
arises through the tanning of the arthropodin vv^hich is converted into
sclerotin which is not soluble in water but is soluble in 5% sodium hy-
droxide at 50°. The tanning of arthropodin to sclerotin results from the
action of a phenolase on a phenol, the resulting quinone combining with
the amino-groups of the arthropodin. The classes Insecta and Crustacea
although both conforming to the general biochemical plan of Arthropods,
differ in the method of hardening of the exocuticle. In the Insecta this
hardening is due to a tanning action, while in Crustacea it is due to cal-
cification.

The Insecta definitely differ from the Crustacea as well as from other
groups of animals in the composition of their blood plasma and particularly
by having a high concentration of free amino acids in this plasma. While,
in other groups, the plasma non-protein nitrogen does not exceed 10 mg
per 100 ml, it may be as high as 300 mg per 100 ml in insects. Other
characteristics of insect blood plasma are the high concentrations of
trehalose, of uric acid and of non-fermentable reducing substances.

In the class Insecta, special biochemical characters of various sub-
divisions may be distinguished. As we have already stated, blood coagula-
tion in insects is of the general type found in Arthropods depending on
changes in special cells called coagulocytes, and the action of their coagulins

332 UNITY AND DIVERSITY IN BIOCHEMISTRY

on the fibrinogen of the plasma. The various visible stages of this coagula-
tion show differences reflecting variations in the underlying chemical
phenomena.

The first type of coagulation is characterized by the development around
the coagulocytes of islands of coagulating material and the extension of the
process to wider areas by the subsequent organization of this coagulum
into a network. In a second type, the coagulocytes extrude threadlike
pseudopodial extensions which form networks of varying complexity and
on which veils of coagulated plasma often appear.

Orthoptera and Dermaptera are uniform in showing coagulation of the
first type. The same type is found among Hemiptera, in the family Nepidae,
the other families of the order lacking the ability to coagulate. The second
type is found in all the Lepidoptera.

Other biochemical characters are peculiar to one or other order of insects.
The plasma inorganic bases of Lepidoptera are characteristic in that there
is a high percentage of magnesium and potassium and a low percentage
of sodium.

Within the order Lepidotera, certain biochemical characters are typical
of certain sub-divisions of the order as has been shown by E. B. Ford in
his extensive studies on the wing pigments of butterflies.

The interest of comparative biochemistry springs from the fact that it is
an extension of, and a support to, taxonomy, and thus it becomes possible
to connect the study of biochemical diversity and the treasure of accumu-
lated knowledge obtained by naturalists over several centuries.

REFERENCES

Fogg, C. E. (1953). The Metabolism of Algae. Methuen, London.
Florkin, Marcel. (1944) L' ivolution biochimique. Masson, Paris.
Florkin, Marcel. (1952). Caracteres biochimiques, des categories supraspecifiques

de la systematique animale. Ann. Soc. Roy. Zool. Belg., 83, 111-130.
Harris, H. (1953). An Introduction to Human Biochemical Genetics. Cambridge

Univ. Press, London.
Haslewood, G. a. D. (1955). Recent development in our knowledge of bile salts.

Physiol Rev., 35, 178-196.
Klein, G. (Editor). Handbuch der Pflanzenanalyse. Springer, Vienna, 1931-1933.
MoLiscH, Hans (1933). Pflanzenchemie und Pflanzenwerzvandschaft. Fischer, Jena.
Williams, R. J. (1956). Biochemical Individuality . Wiley, New York.

CHAPTER IV

BIOCHEMICAL EVOLUTION

I. DEFINITION

BiOCREMiCAL evolution should not be considered as a fairy tale for
grown-up biologists, but simply as a search for a natural order among the
many diverse biochemical characters manifested in organisms. It simply
means that when we notice a biochemical change along a branch of the
taxonomist's phylogenic tree we shall call it a fact of biochemical evolution,
meaning that in the two cases considered one is more specialized and the
other more primitive. The identification of the primitive and of the
specialized is decided, to start with, by the standards of the taxonomist,
though not without taking into account the whole of biological knowledge.
Biochemical evolution, as defined above, will be manifest by a change in the
molecular structure of an organic compound or by changes in enzyme
systems, these changes being either complications or simplifications as the
case may be. There may also be changes in the steady states we commonly
call the composition of the organism.

II. EVOLUTION OF BIOCHEMICAL CONSTITUENTS

Before considering the evolution of organisms from the biochemical
angle, we must define a number of useful concepts.

Isology. The term isologues is applied to biochemical compounds, mole-
cules or macromolecules, which show signs of chemical kinship. The
cytochromes, peroxidase, catalase, haemoglobin, chlorocruorin, are iso-
logues because they are haem derivatives. The maximum degree of isology
occurs in the case of haemoglobins of two dogs from the same litter, it is
less if we consider the haemoglobin of a dog and that of a jackal, still less if
we consider that of a dog and that of a horse. In every case the haem,
protohaem, is identical, the degree of isology depending on the nature of
the globin. The degree of isology is still less in the case of a haemoglobin
and of a catalase, the protein portions of the molecule being much more
unlike than in the case of two haemoglobins. Another example of a lower
degree of isology is provided by haemoglobin and chlorocruorin in which
the haems are different. It is clear that there is a whole range of degrees of
isology, always definable in organic chemical terms, for isology is a purely
chemical concept.

333

334 UNITY AND DIVERSITY IN BIOCHEMISTRY

Analogy. This term is applied to chemical units playing the same role
in a biochemical system. Phosphocreatine and phosphoarginine in the
muscles of mammals and crustaceans respectively, are analogues. Bio-
chemical units may be both isologues and analogues, as is the case for a
blood haemoglobin and a blood chlorocruorin : both serve as oxygen
carriers. But this is not necessarily so : a blood haemoglobin and a
haemocyanin are analogues, but they are not isologues.

When we have information about the relative positions in the classifica-
tion of a number of species, and about the isologic relations of one or other
of their biochemical constituents, then it becomes possible to draw con-
clusions about the biochemical evolution of these constituents. Consider,
for example, chlorocruorin, the oxygen carrier in the Chlorhemians, in the
Sabellidae and the Serpulidae. This carrier is present in the blood of
three families of polychete Annelids. The Chlorhemians are Spionids, a
classification which groups among the sedentary polychete Annelids, which
are descended from the errant polychete Annelids, those forms whose
preoral lobe is not sunk into the first segment of the metasome, and which
feed on floating plankton gathered by means of posterior antennae in the
form of long palps bearing a ciliated gutter. They live in sand or mud and
secrete a membranous tube covered with a fine layer of slime. The
Chlorhemians are Spionids which have lost the dissepiments and even the
external segmentation. Their blood is green, and their palps are folded
forward. Related to the Spionids are the cryptocephalic Annelids, having
a preoral lobe sunk into the first segment of the trunk, but possessing
furrowed appendages like those of the Spionids. Sedentary and tubicolous,
the Cryptocephalae comprise two sub-divisions, the Sabellariides, which
although sedentary and microphagic, have retained an uneven number
of antennae, and the Sabelliforms which have even antennae and palps
forming a multicoloured corolla. The Sabelliforms are divided into the
Sabellidae, having a mucous tube, membranous or cornified, and the
Serpulidae, possessing a calcareous tube.

The blood of Spionids other than the Chlorhemians is coloured red by
haemoglobin. When we come to the Sabellariides, one of the genera of
which is Sabellaria, their blood is charged with haemoglobin. In the
Sabelliforms, sometimes called the Serpuliforms, chlorocruorin is the
characteristic blood pigment. All the Sabellides so far studied contain it.
Among the Serpulides blood of species in the genus Serpula contain
both chlorocruorin and haemoglobin and in the genus Spirorbis, one
species, S. borealis has a blood coloured by chlorocruorin, another,
S. corrugatus contains haemoglobin and a third, S. militaris has a colourless
blood. H. M. Fox (1949) did not find chlorocruorin in the tissues or in the
coelomic fluid of the forms having chlorocruorin in the blood. No doubt,
in those forms which contain it, the synthesis of chlorocruorin is a variant of

BIOCHEMICAL EVOLUTION

335

haemoglobin synthesis as it was present in their Annelid ancestors possess-
ing this synthetic mechanism. Also too, chlorocruorin is a close isologue
of Annelid haemoglobin and possesses many similar properties. The haem
of chlorocruorin, or chlorocruorohaem, differs from protoporphyrin in
only a small detail, the oxidation of vinyl group 2. As for the protein
portion, it is very similar to that found in Annelid haemoglobin as the data
assembled in the following table show :

Table XXIII

Iso-

Molecular

Containing

amino acids

electric

weight X

Cystine

Arginine

Histidine

Lysine

point

17000 ( + )

%

/o

%

%

Haemoglobin of

horse

6-78

4

0-74

3-57

8-13

8-31

Haemoglobin of

lombric

5-28

192

1-41

10.07

4-68

1-73

Haemoglobin of

arenicola

4-76

192

4-08

10-04

4-03

1-85

Chlorocruorin of

4-3

192

1-64

9-64

2-38

3-64

Spirographis

In the case of chlorocruorin, we have a chemical entity isologous to
haemoglobin and present in classes of our systematic classification and the
comparative morphology of these classes shows their phylogenic relation
to other classes the blood of whose members contains haemoglobin. Here
we can speak of evolution of a biochemical component. In a case of this
type there is not only isology, but phylogenic isology, or to use a term
proposed by Lankester, homogeny.

Biochemical Parallelism

When isologous biochemical constituents are present in categories of
the systematic classification not phylogenically related, we say that there is
parallelism. The occurrence of haemoglobin in the Molluscs and in the
Echinoderms is an example of parallelism. In fact, parallelisms are evidence
for the unity of the general biochemical plan on which organisms are
constructed.

REFERENCES

Pedersen, K. O. (1933). Koll. Z., 63, 268.

Roche, J. and Jean, G. (1954). Bull. Soc. Chim. Biol., 16, 769; and Roche, J. and

MouRGUE, M. (1941), Bull. Soc. Chim. Biol, 23, 1329.
SvEDBERG, T. (1933). X Biol. Chem., 103, 311.
SvEDBERG, T. (1939), Proc. Roy. Soc, Ser. B., Ill, 1.
VAN Slyke, D. D., Hastings, A. B., Heidelberger, M. and Neill, J. M., (1922).

y. Biol. Chem., 54, 81.

336 UNITY AND DIVERSITY IN BIOCHEMISTRY

Biochemical Convergence

When biochemical constituents are analogues without being isologues,
as is the case for haemoglobin and haemocyanin, we say that there is bio-
chemical convergence.

Heterotnorphic Evolution

The phylogenic isology shown by the chlorocruorins and the haemo-
globins of Annelids gives us an example of what can be called hetero-
morphic evolution, revealed by the acquisition of a modified constituent
of a less complete isology. The haemoglobins with a strong affinity for
oxygen appear to be more primitive than haemoglobins of weak affinity,
and here we are dealing with a heteromorphic evolution of their protein
component (molecule evolution).

In the higher animals, the dissociation of haemoglobin varies with its
degree of oxygenation. The oxygenation of haemoglobin displaces the
isoelectric point : in the horse for example, the isoelectric point of reduced
haemoglobin is pH 6-78, and that of oxyhaemoglobin is pH 6-65. At the
isoelectric pH, haemoglobin fixes small but equivalent amounts of acids and
bases, but since the pH in red blood cells is on the alkaline side of the
isoelectric point, the haemoglobin is present in combination with bases
as the salt.

At the pH of blood, haemoglobin behaves like a polyvalent acid with at
least five acid groups per atom of iron.

Horse oxyhaemoglobin whose isoelectric point is pH 6-65, contains,
among others, an acid function (or groups of functions) which is weakly
dissociated with a pK of 6-16. In the corpuscles of oxygenated horse
blood the pH is 7-1 and this function for the most part is saturated with
bases. The haemoglobin has a less acid character since its isoelectric point
is at pH 6-8. This modification of the isoelectric point by a change in the
oxygenation is due to a considerable decrease in the dissociation of the
acid groups in the region of the oxygen-binding group, as we have des-
cribed above. Their pK changes from 6-16 to 7-80. When such a change
occurs in a medium whose pH varies hardly at all, there is a resulting
liberation of the bases fixed to the acid function. The observation that
reduction of blood raises the curve of CO2 absorption is known as the
"Haldane effect". It appears to be a character in the evolution of haemo-
globin due to a modification of the protein moiety and consequently is an
example of heteromorphic evolution. In fact the effect is not observed in
the blood of the ray, of Mustelus cants or of Urechis all of which contain
haemoglobins the dissociation of which does not vary with the degree of
oxygenation.

The molecular evolution of proteins, as the above examples indicate,
is evidently an essential facet of the evolution of organisms. Recent work

BIOCHEMICAL EVOLUTION

337

has enabled us to obtain a clearer view of the heteromorphic aspects of
proteins which are more or less isologous and new horizons have been
opened up.

The molecular weights of Vertebrate haemoglobins are situated around
65,000-68,000. The molecules are apparently made up of two systems
of three peptide chains. The N-terminal amino acid sequence of a number
of haemoglobins is given in Table XXIV. There would appear to be a com-
mon basic chain in all cases.

Table XXIV

(Osawa and Satake 1955)

Species

Horse, pig

Dog

Bull, goat, sheep

Cobaya

Rabbit, Snake

Hen

N-Terminal sequences

Val. Leu.
Val. Leu.
Val. Leu.
Val. Leu.
Val. Leu.
Val. Leu.

Val. Gly.

Val Glu. (Leu.)

Val. Gly.

Val. Asp.

Met. Gly.

Val. Ser.

Val. Asp.

Val. Gly.

It would be extremely satisfying to be able to follow in homologous
tissues, in every phyletic line, the stages in the biochemical evolution of
each type of macromolecule in the cell. But our knowledge of the field of
comparative biochemistry is still too sparse for such a task. On the other
hand in the field of phylogeny, the gaps left in the explanation of the kinship
between one class and the next are enormous, and living beings as we know
them appear to possess such a continuity that certain writers have been
led to deny the idea of evolution altogether. Frequently biochemical
constituents are found in one group without it being possible to study
biochemically a group that can be said, with certainty, to be the immediate
predecessor in the phyletic series. The general outlines of phylogeny can
however serve to define the more or less primitive or specialized character
of a biochemical constituent.

Let us consider, for instance, the distribution of porphyropsin and
rhodopsin (p. 290) in the visual organs of animals. A diagram systematizing
the observations of different workers and particularly of George Wald and
his school (Fig. 90b) shows, in the phylogenic series represented here, a
definite chemical evolution in which porphyropsin appears to be more
primitive than rhodopsin. At the present time it is generally agreed that
Vertebrates originated in fresh water, and it is conceivable therefore that
the early Vertebrates already possessed the porphyropsin system. This,
of course, is not a matter open to direct investigation but we may notice
that the African Lungfish Protopterus, which is believed to have descended

338

UNITY AND DrVERSITY IN BIOCHEMISTRY

Table XXV

(W. Bergmann, 1949)

Distribution of the unsaponifiable portion of the lipids of animals

% of unsaponifiable in

Phyla or classes

relation to the total
lipids

Number of examples

Protozoa

35

2

Porifera

37

45

Coelenterata

35

16

Nemathelminthes

25

1

Annelida

22

7

Crustacea

16

14

Myriapoda

21

1

Insecta

7

31

MoUusca (marine)

13

18

Echinodermata

19

10

Chordata

1-2

50

Table XXVI

(W. Bergmann, 1949, 1952)

Phyla and Classes

Distribution of sterols in animals

Principal sterols

Porifera

cholesterol, cholestanol, clionasterol, poriferasterol,

chalinasterol, neospongosterol, chondrillasterol,

halicionasterol, aptostanol and others.

Coelenterata

cholesterol, clionasterol, chalinasterol, palysterol and

others

Annelida

cholesterol

Arthropoda

cholesterol

MoUusca

Pelerypoda

cholinasterol, brassicasterol, corbisterol, cholesterol

and others.

Castropoda

cholesterol

Cephalopoda

cholesterol

Echinodermata

Asteroidea

stellasterols

Holothuroidea

stellasterols

Echinoidea

cholesterol

Protochordata

cholesterol

Chordata

cholesterol

BIOCHEMICAL EVOLUTION 339

Marine fishes (Ai) Land vertebrates (Ai)

Catadromous fishes (Ai > A2)

\
Anadromous fishes (A2 > Ai)

Amphibia (A2 and Ai)

Freshwater fishes (A2)
Lampreys (A2 and Ai)

Ancestral vertebrates {Ao})

Fig. 90b — (Wald). Distribution of vitamins A^ and A2 in vertebrate retinas.

from a continuous fine of fresh-water ancestors, possesses the porphyropsin
system. The use of vitamin Ag in the retina appears to be universal in
fresh-water Vertebrates and to extend as far back to the origin of the
Vertebrates as it is possible to penetrate.

W. Bergmann has emphasized that a marked difference exists, (which
can be seen by reference to Table XXV) in the material extractable with
fat-solvents when we compare the Vertebrates and the Invertebrates :
there is a greater proportion of unsaponifiable matter in the latter.

Moreover, Bergmann noted that if we consider the distribution of
sterols in animals, the greatest diversity of sterols is found in the most
primitive groups while in the most highly specialized, cholesterol is almost
the only one identified (Table XXVI).

Among the sterols of the least specialized animals we find Cgg or C29
compounds. Sterols of this type differ in the type and degree of unsatura-
tion, and in the nature of the radical attached at C-24, etc. (Bergmann,
1952). On the other hand, from a series of studies by Haslewood and
co-workers, we see that in teleost fishes or Elasmobranchs, Amphibians,
crocodiles and alligators, in a lizard, in Chelonians (turtles and tortoises)
and in some birds, the bile salts contain C27, C28 (or possibly C29) alcohols
and acids whilst in snakes and mammals, substances of this type are not
found. But the presence of C24 bile acids has been demonstrated in snakes,
teleost fishes, mammals and birds, but not in Elasmobranchs, Batracians
or reptiles such as the Crocodilians and the Chelonians.

In general, the C27, C28 and possibly the C29 bile salts go with a more
primitive phylogenic status than with that of the more specialized animals
which have C24 bile salts.

340 UNITY AND DIVERSITY IN BIOCHEMISTRY

Another example connected with the general idea of phylogeny is one
cited by Comfort, who pointed out that the depot uroporphyrin of the shelf
occurs chiefly in the less specialized Archaeogastropoda. And it is possible
to quote many other examples,

III. EVOLUTION OF BIOCHEMICAL SYSTEMS

In the preceding, we have considered the constituents of an organism,
i.e. from the point of view of the macromolecules and organic molecules
contained in it. But these compounds are evidently derived from the opera-
tion of biosynthetic systems, and thus, a heteromorphic evolution, such as
the replacement of a haemoglobin by a chlorocruorin, occurs in the bio-
synthetic mechanism which produces that constituent. Hence this change
is in an enzyme system, i.e. in a system of macromolecules the nature of
which is controlled in every case by a gene, itself being on occasion the
object of a heteromorphic evolution and of a reduction in the isology of its
nucleic acids with those of its forbears. If we grant that the photosynthetic
pathway (see Part Six) is a metabolic variant of the hexosemonophosphate
shunt, then photosynthesis regarded as a reduction of COg will be
carried out by a system more specialized than the hexosemonophosphate
shunt. On the other hand, if it is true that at the beginning the biosphere
was lacking in COg, photosynthesis could only appear after the liberation
of this substance from volcanoes and primitive forms of metabolism.
If the presence of oxygen in the terrestrial atmosphere has depended
on photosynthesis then that part of the overall metabolic schemes which
concerns respiration is biochemically more specialized than that con-
taining glycolysis and the hexosemonophosphate shunt. But all this
lies in the domain of prehistoric biochemistry and consequently is
of a highly speculative nature. In the biosphere at the present time we
have before us what survives of many diversifications of the general plan
of cellular biochemistry described in Part III, and evidently what remains
has been preceded by more primitive systems which today have dis-
ppeared.

(a) Specialization by Quantitative or Topographic Modifications

Extracellular digestion compared to intracellular digestion implies that
specialization has occurred in the sense of a relatively great biosynthesis of
enzymes secreted into the lumen of the digestive tube and constantly
renewed. Intracellular digestion is the primitive form. It is the only form
of digestion in the Spongiae.

As Yonge has emphasized, an example that demonstrates very well the
relation between the system of intracellular digestion and that of extra-
cellular digestion is the Molluscs : among them we find all stages between
an almost complete intracellular digestion and a totally extracellular

BIOCHEMICAL EVOLUTION 341

digestion based on the secretion of enzymes into the lumen of the digestive
tube. In general, Lamellibranchs feed by a ciliary mechanism which is
responsible for the collection of fine particles, mainly of phytoplankton.
The only extracellular phase of digestion they have is in the action of an
amylase, all the other enzymes acting intracellularly. Among the herbi-
vorous Gastropods (the Pulmonata excepted), like Yonge we can distin-
guish two groups : those possessing a crystalline style and those which do
not. In the former, as for example the Streptoneura, conditions are very
similar to those found in the Lamellibranchs, amylase being the only
extracellular enzyme, the digestive diverticulae acting as organs of absorp-
tion and intracellular digestion, but never of secretion. The second group
of herbivorous Gastropods, those not possessing a crj^stalline style, as is
the case for the Tectibranchs and the Nudibranchs, show considerable
diversity and in certain cases there is a proteinase in the juice in the
digestive tube. When we come to the carnivorous Gastropods such as
Murex, a proteinase actively secreted by the digestive diverticulae is
always found in the digestive tube. Among them, too, there is also
intracellular digestion. In addition, the salivary glands secrete amylase.
Among the Pulmonata, such as the snail, the hydrolytic processes are
almost completely extracellular. Only protein hydrolysis is intracellular.
In the Cephalopods, digestion is exclusively extracellular and intra-
cellular digestion has disappeared.

(b) Specialisation by Acquisition of a New Constituent as a
Result of Molecular Evolution

During the course of the evolution of the cells that contain it, an enzyme
system may become the object of a specialization of a new type. Snakes,
for example, do not mix digestive secretions with their prey by a process
of mastication. They swallow their prey after having injected it with a
secretion which initiates hydrolysis. In the least specialized form, for
example in Colubridae opisthoglyphae, a simple secretory tooth appears
at the rear of the upper jaw and serves for the injection of a secretion whose
function is purely digestive. In more specialized forms, this organ,
following a decrease in length of the maxilla, approaches the anterior part
of the buccal cavity and becomes an aggressive and defensive organ, as is
the case in Colubridae proteroglyphae and even more so in the Viperidae.

The digestive origin of the secretion is further borne out by the presence
in snake venoms of such hydrolases as proteases, peptidases, phosphatases,
sterases, and lecithinases. The new specialization expresses itself by the
presence of hyaluronidase, assuring the diffusion of the venom, and by the
presence of substances of high toxicity (see Zeller, 1948).

Another example of modification of an old system by the addition of a
new component is the urea-synthesizing system in the hepatic parenchyma

342

UNITY AND DIVERSITY IN BIOCHEMISTRY

HN CO

I I H
OC C N

/
HN C NH

CO

uric acid

{uricase)

I
HjN

I O H

OC C — N

HN— C N

/

CO

H H

allantoine

I.
(allantoinase)

I
HgN NH2

I I

OC COOH CO

I I I

HN— C NH

H

allantoic acid

i .
(allantoicase)

COOH NHj

/ +20=C

NH,

H

Urea

I
(urease)

I
2NH3 + CO2

Ammonia

Fig. 91 — The uricolytic enzyme system.

BIOCHEMICAL EVOLUTION

343

of ureotelic Vertebrates (p. 309). The new function appears as the result
of a speciaHzation in which arginase has been added to the enzyme system
bringing about the synthesis of arginine (p. 244).

(c) Specialization by Loss of Constituents

An enzyme system may be specialized not only by the acquisition of
new enzymes, but also by the loss of certain existing ones. An example is
the enzyme system of uricolysis. The most complete form of this system is
found in marine Crustaceans and is shown in Fig. 91.

Most Insects only carry uricolysis to the stage of uric acid. The form
of the uricolytic system in Insects compared to the most primitive form of
the Crustaceans, is characterized by the disappearance of urease, allan-
toicase, allantoinase and uricase. The enzyme system for purine break-
down consists of uricase, allantoinase and allantoicase in the Batracians, it
consists of only uricase in the Mammals, with the exception of the
Primates who have lost the complete system of enzymes as likewise have
the terrestrial Reptiles and Birds.

{d) Specialization by Introduction of a Constituent of a Primitive System

into a more Modern System

Rhodopsfn

Vitamin Ax + Opsin"^

Vitamin Aj of

the pigmented

epithelium and

of the circulation

DPNH -i- H t- \

/ Systems of \
y oxidation J

DPNH -'- H+

\^ Light

\

^ Lumi-rhodopsin

I -20='C.

Meta-rhodopsin

Retincne -p Opsin

Fig. 92 (G. Wald). — The rhodopsin system.

A frequent form of evolution of enzyme systems and their associated
systems (substrates, enzymes, coenzymes, hormonal regulators, etc.) is
by the introduction of one or several of their components into a more
specialized system. We have already noted (p. 290) the more specialized
nature of rhodopsin, present in the retina of salt-water fish, reptiles, birds
and mammals, compared to the more primitive porphyropsin of fresh- water

344 UNITY AND DIVERSITY IN BIOCHEMISTRY

Vertebrates. Rhodopsin is a derivative of vitamin Aj and porphyropsin
is a derivative of vitamin Ag. The visual function of the A vitamins
is the only one it has been possible to demonstrate in animals other than
the mammals and birds. In the two latter, it plays the additional role of a
vitamin essential for the normal function of epithelial tissue. This last fact
shows us the development of a new biochemical system in which has been
inserted a biochemical component already utilized in another system. The
photoreceptor system of the rods in the retina of animals having a differen-
tiated eye furnishes us with another example of this type of evolution in an
enzyme system. Figure 92 indicates the changes taking place during
photoreception in the rhodopsin of the eye. Retinene is produced by the
reduction of vitamin A^. The enzyme catalysing the transformation was
first called retinene-reductase. We know today that it is alcohol-
dehydrogenase (p. 162) as has been shown by Bliss. This universally
distributed enzyme is found to have been inserted here into a new system
that is extremely specialized.

The mechanisms of hormonal regulation provide us with many instances
of insertions into new systems. The secretion of milk, due to the bio-
chemical differentiation of one type of Mammalian cell (p. 306) is provoked
and controlled by prolactin, resulting from the biochemical specialization
of another type of cell, the adenohypophysis. But prolactin is secreted by
the adenohypophysis of fish, amphibians and reptiles. Its intervention
in the secretion of milk in mammals is thus an insertion into a new bio-
chemical system.

Another example of the same type is the action of pitocin on the
mammalian uterus. This hormone is present in all Vertebrates and
acts in the control of water metabolism. Its action on mammalian uterus
demonstrates its insertion into a more specialized system.

(e) Specialization of a Primitive Biochemical System by the Introduction
of a Constituent of another Primitive System

One of the important aspects of the biochemical evolution of Vertebrates
has been the acquisition by the cells of the mesoderm of enzyme systems
for the biosynthesis of new types of steroid (heteromorphic evolution).
One of the physiological effects of this evolution is the ionic regulation
brought about by the action of the corticosteroid hormones at the urinary
tube. In the Amphibiae this action is established in conjunction with a
pre-existing system, that of the regulation controlled by the adeno-
hypophysis.

The adaptation to terrestrial life in certain amphibians, such as the toad,
in fact depends on the ability to reabsorb water controlled by the active
principles of the hypophysis, and these substances are fundamental con-
stituents in these animals (see Jones, 1957).

BIOCHEMICAL EVOLUTION 345

In many cases the biochemical systems characteristic of such and such a
cellular differentiation appear to be biochemical inventions the past
history of which we are so far unable to reconstruct. Such systems are to
be found among the many different biochemical modifications in Verte-
brates, for example, the complex enzyme systems for the degradation of
haemoglobin present in histiocytes, the conversion of cholesterol to bile
in the cells of the hepatic parenchyma, etc. The fact that we do not know
the systems preceding them in phylogeny does not invalidate the fact that
these biochemical inventions have evolved.

Scanty as it is at the moment, our knowledge of the biochemical diversity
of organisms has indicated that more detailed studies could tell us much
about the methods according to which the extension of the biosphere has
been accomplished along the lines of biochemical evolution, the bio-
chemical diversity being, as we shall see in Part VI, as essential as the
biochemical unity, for the maintenance of the metabolism of the whole
biosphere in extension.

REFERENCES

Anfinsen, C. B. (1959) The Molecular Basis of Evolution, John Wiley, New

York.
Bergmann, W. (1949). Comparative biochemical studies on the lipids of marine

invertebrates, with special reference to the sterols. Sears Found. J. Marine

Res.,%,Ul-\16.
Bergmann, W. (1952). Sterols. Progr. Chem. of Fats and other Lipids, 1, 18-69.
Florkin, M. (1944). U evolution biochimique. Masson, Paris.
Jones, I. C. (1957). The Adrenal Cortex. Cambridge Univ. Press.
Wald, G. 1951. The chemistry of rod in Fish. Science 113, 287-291.
YoNGE, C. M. (1937). Evolution and adaptation in the digestive system of Metazoa.

Biol. Rev., 12, 87-115.
Zeller, E. a. 1948. Enzymes of snake venoms and their biological significance.

Advanc. EnzymoL, 8, 459-495.

PART SIX
THE METABOLISM OF THE BIOSPHERE

INTRODUCTION

The series of priming reactions and synthetic mechanisms described in
Part Three of this book give some idea of the chemical processes occurring
in the biosphere. Superimposed on this general background there are
many variations, simplifications or amplifications arising from the differen-
tiation, adaptation and evolution of both the cells and the organism.
Without them life would become extinct.

Each organism is a link in a food chain whose beginning varies according
to the particular association of living creatures. In a pond, bacteria and
other micro-organisms are at the beginning of the chain. Crustaceans
feed on the micro-organisms and themselves act as food for aquatic insects
that, in turn, are eaten by fish. The dead bodies of the latter serve to
nourish bacteria. Animals feeding on plants are the prey of carnivorous
animals, that are themselves eaten by other carnivores. This concept of
food chains demonstrates how the macromolecules in the cells of an
organism can serve as food to other organisms which begin by hydrolysing
them with the aid of the arsenal of hydrolases so liberally distributed
throughout the biosphere. In this way one part of the biosphere serves to
feed the other part. However, if each portion of the biosphere was
nourished solely by the consumption of another portion, life would be
progressively stifled and would disappear in a very short time. In the same
way as for the metabolism of a single organism the metabolism of the
biosphere implies an entry and an exit of matter and energy. This
continuous exchange with the surroundings constitutes the general
metabolism of the biosphere which is dealt with in Part Six.

349

CHAPTER I

ENTRY INTO THE BIOSPHERE

I. CARBON AND ENERGY
In addition to the priming and biosynthetic reactions described in Part
Three we mast consider the entry of energy and matter which occurs in
certain regions of the biosphere. As we have pointed out, the priming
reactions constitute a chemical machine which forms, at the expense of the
chemical energy of nutrient molecules, energy-rich bonds of ATP, packets
of energy which can be utilized for biosynthesis. Also, during the function-
ing of this chemical m.achine construction materials are produced which
can be employed for biosynthetic purposes.

However organisms exist which are capable, by chemical mechanisms of
their own, of introducing into their metabolism a supply of external
energy, either chemical or electromagnetic in nature. Traditionally, the
name chimiosynthesis is given to the synthesis of carbohydrates from
CO2 and chemical energy. In this sense, all organisms are chimiosynthetic.
But, certain micro-organisms, during the synthesis of carbohydrates,
introduce energy derived from the oxidation by oxygen (auto-oxidation) of
a constituent of the external medium. Other organisms are capable of
carr}dng out photosynthesis, i.e. synthesis of carbohydrates using electro-
magnetic energy derived from light. So, by the terms chimiosynthesis and
photosynthesis we understand that sugars are synthesized. The term
photometabolism refers to other types of metabolic action accomplished
by light.

There are some micro-organisms who obtain all their energy and material
from outside the biosphere. These are the autotrophes. They build up
all their constituent organic material from COg, HgO and other inorganic
substances like ammonia, sulphates and phosphates. For energy they use
that derived from the oxidation of substances in the surrounding medium.
Photosynthesis, another form of autotrophism, occurs in certain bacteria,
in algae, in diatoms, in green plants, etc.

A. Autotrophic Bacteria

These organisms do not obtain their nutrient from some other region
of the biosphere: the flow of energy and matter through them is derived
from the inorganic world. Examples are: the nitrous and nitric bacteria
and the colourless sulphur bacteria.

351

352 UNITY AND DIVERSITY IN BIOCHEMISTRY

The autotrophic bacteria are chimiosynthetic, that is, they synthesize
sugars using the energy of oxidation of a constituent of the surrounding
medium.

(a) Nitrous Bacteria

These bacteria accomplish a nitrosation (ammonium sahs -> nitrites).
These bacteria are numerous and belong to the genera Nitrosomonas
(aerobic, widely distributed in the soil of Europe and Asia, ovoid or
spherical in shape, able to move in a liquid habitat by means of their
flagellae they group themselves in mucilaginous zoogloeae) and Nitro-
sococcus, (in American soil, always non-mobile). The chimiosynthesis of
carbohydrates in the nitrous bacteria consists of a transfer of hydrogen
from ammonia to COg:

2NH3 + 2O2 -> 2NO2H + 2H2

2H2 + CO2 -> CH2O + H2O

{b) Nitric Bacteria

These oxidize nitrites to nitrates thus performing a nitration. Among
the nitric bacteria are the Nitrobacter which are bacilliform, non-mobile,
and aerobic (Fig. 93).

^^ 0. ^N

/ ^.s. ^^ n

Fig. 93 (Winogradsky) — Nitrobacter

{c) Colourless Sulphur Bacteria

Certain of these are aerobic, others anaerobic. Among the aerobes are
Thiobacillus thioparus and Thiobacillus thiooxydans. The former is present
in soil in the form of small rods. It multiplies when the medium is neutral.
It oxidizes thiosulphates (hyposulphites) or sulphides to sulphate and
sometimes sulphur is deposited inside the cell.

2Na2S203 + O2 -> 2Na2S04 + 2S

Thiobacillus thiooxydans is found in the soil in the vicinity of sulphur
deposits. It produces large amounts of H2SO4 and only grows under acid
conditions (pH 8 = 2-0-3 -0). It oxidizes sulphur or thiosulphate to
sulphuric acid:

2S + 3O2 + 2H2O -> 2H2SO4

Na2S203 + H2O + 2O2 -> Na2S04 + H2SO4

ENTRY INTO THE BIOSPHERE 353

Certain other autotrophes which oxidize sulphur are anaerobic. This is
the case with Thiobacillm denitrifians which oxidizes sulphur, HgS,
NagSgOg (with formation of H2SO4), at the expense of oxygen derived
from nitrates. It is widely distributed, being present in soils, water and
muds.

SNagSgOg + 8KNO3 + 2NaHC0g ^ eNagSO^ + 4K2SO4 +

4N2 + 2CO2 + H2O

In addition to the simple bacteria we have just considered, there are
some morphologically more complex, such as Thiothrix and Beggiatoa
who oxidize H2S to H2SO4 using part of the energy from this reaction to
synthesize carbon chains. They are aerobes containing particles of
sulphur in their cells. In addition to COg and oxygen they require the
presence of H2S. As long as H2S is present, the sulphur particles remain
in the cells. In the absence of HgS, these bacteria can use their reserve of
sulphur granules. When this reserve is exhausted, they die. The energy-
yielding reactions are as follows:

H2S + O -> H2O + S

2S + 3O2 + 2H2O -^ 2H2SO4

{d) The Mechanism of Autotrophy

The autotrophic bacteria pose the major problem of how the energy
produced by an oxidation reaction is transferred and used for the synthesis
of a sugar. In a general way the process can be represented as occurring
in the following stages:

1. Activation by an enzyme of the oxidizable substrate (a thiosulphate
dehydrogenase in Thiobacillus, an ammonium dehydrogenase in Nitro-
somonas).

2. Oxidation by the cytochrome-cytochrome oxidase system.

3. Formation, during this oxidation, of energy-rich phosphate bonds.

4. Fixation of COg by conversion to a carboxyl group, in the usual way.

5. Formation of a carboxyl phosphate.

6. Reduction of the latter by the enzyme of reaction 1, liberation of
phosphate.

XCO— O - H2PO3 + RHo ^ XCHO + H3PO4 + R

However the above is almost entirely hypothetical.

B. Photosynthesis in the Green Algae and Green Plants

Photosynthesis by green algae and green plants is the major means of
introducing carbon into the biosphere. The annual production of organic
substances by photosynthesis in the biosphere is 2000 times greater than

354 UNITY AND DIVERSITY IN BIOCHEMISTRY

the annual production of steel throughout the world, which is 100 million
tons per annum.

According to Loomis, photosynthesis produces 270,000,000,000 tons of
glucose per annum and consumes 396,000,000,000 tons of carbon dioxide.
Statistically, over 2000 years all the COg molecules in the air will have been
incorporated into the biosphere at some time or other.

In plants and algae, photosynthesis takes place in cytoplasmic inclusions,
the chloroplasts.

(a) The Chloroplasts

The chloroplasts in green algae and plants are the seat of photosynthessi
and producers of glucose- 1 -P. They are disc-shaped particles 3 to 10 (x in
diameter and 1 to 2 [i, in thickness. It is possible to isolate them from
leaves and show that they are bounded by a definite membrane which is
semi-permeable. The chloroplasts contain a number of gratia whose
diameter varies, according to the type of cell, from 0-2 to 2 [x. A chloroplast
contains from 10 to 100 grana imbedded in a protein matrix. The electron
microscope reveals that they have a laminar structure. They contain 33 to
50% protein and also contain lipides. The chloroplasts are auto-
reproductive and can divide.

There are two types of pigments in the chloroplasts, chlorophyll and
carotenoids. Besides chlorophyll, the chloroplasts of the red algae contain
other tetrapyrrole pigments which are active in photosynthesis, these are
the phycobilins. The leaves of green plants often contain in the vacuole
(i.e. outside the cytoplasm), pigments such as the anthocyanins which do
not play any part in photosynthesis.

(b) History of the Ideas Relative to Photosynthesis

It was not until 1727 that Stephen Hales expressed doubts about the
correctness of the then current view that plants obtained all their nourish-
ment from the soil. Hales suggested that air and light also played a part.
The next step was due to Priestley who showed, in 1771, that green plants
gave out oxygen. The following year Ingenhouz demonstrated the part
played by light in this phenomenon. In 1782 Senebier discovered that
CO2 was also concerned in the production, in light, of oxygen by green
plants. In 1804 quantitative methods of study were applied by Saussure.
He showed that the weight gained by a plant over a given period during
photosynthesis, plus the weight o:^ oxygen liberated, is greater than the
weight of CO2 taken in. He suggested that water played a part. In 1941
Van Niel showed that the photosynthetic purple algae, the Thiorhodaceae,
can multiply in an anaerobic inorganic medium provided that the medium
contains HgS. It was also shown that the amount of growth is proportional
to the HgS concentration. So photosynthesis must be considered as being

ENTRY INTO THE BIOSPHERE 355

linked to an oxido-reduction reaction, and in Van Niel's theory, which is
generally accepted today, the hydrogen donor is always HgO. Up to about
1935 studies of photosynthesis were concentrated on the effect of various
factors on the rate of photosynthesis in the living plant. Much work has
been done from this physiological aspect, the main result being only to
underline the mystery of photosynthesis.

With the advent of methods employing radioactive isotopes and bio-
chemical methods for the study of isolated systems, it became possible to
penetrate into the intimate mechanism of photosynthesis. In 1940 the
reaction of Hill (p. 357) was announced and the first experiment using
isotopes (by Ruben, Kamen and Hassid, using ^^C, the only available
carbon isotope at that time) was carried out. The presence of magnesium
in chlorophyll had led, by a process of reasoning on chemical grounds,
to the idea that the first step in biosynthesis was a photochemical reaction
involving chlorophyll and COg. This idea stemmed from three observa-
tions: the presence of magnesium in chlorophyll, the reduction of COg
to formaldehyde by mietallic magnesium in acid solution, and the feeble
biochemical reactivity of COg.

Today, we know that on the contrary COg is a metabolite of general
importance. Facts obtained since 1940 lead us to beheve that photo-
synthesis is a process in which the photochemical reaction is a photolysis
of water preparatory to the transfer of hydrogen.

According to the ideas of Calvin, the priming reaction for photosyn-
thesis, that is the transformation of water under the influence of light,
furnishes much TPNH and ATP from the electromagnetic energy of the
light. Also pyruvic acid is diverted to the photosynthetic cycle as a result
of the presence of thioctic acid in its dithiol form.

By contrast, in the dark, oxidation takes place and the disulphide form
is re-established; the carbon then takes a path required by respiration.

(c) The Initial Stage of Photosynthesis

The first stage is a process of quantum absorption converting water into
a reducing agent and an oxidizing agent.

H2O -^ [H] + [OH]

In green plants oxygen is liberated during this first stage. In photo-
trophic bacteria, the oxidizing radical [OH] must be reduced by a hydrogen
donor which is specific in each case.

The second stage is a reduction

CO2 + [H] -> (CH^O)^

356

UNITY AND DIVERSITY IN BIOCHEMISTRY

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ENTRY INTO THE BIOSPHERE

357

This division into two stages is confirmed by the Hill reaction, i.e. the
production of oxygen by isolated chloroplasts in the presence of a hydrogen
acceptor such as benzoquinone or 2,6-dichlorophenol. By means of
labelled oxygen it has been proved that the oxygen liberated in the Hill
reaction is derived from the water.

Fig. 95. — Racker's reaction scheme showing the formation of a molecule of triosephosphate
from three molecules of CO2 in the pentose phosphate cycle acting as a reductive cycle

during photosynthesis.

{d) The Reduction of Carbon Dioxide
Secotid Stage of Photosynthesis

Carbon dioxide is first fixed in a carboxyl group, in the general way,
the reverse of decarboxylation. This CO2 appears in the carboxyl of
3-phosphoglyceric acid. Although the role of 3-phosphoglyceric acid is
usually admitted, there is still much discussion as to the nature of the
substance which first combines with the COg.

Calvin and his school believe that ribulose diphosphate is the immediate
precursor of 3-phosphoglyceric acid. Thus the photosynthetic cycle
would begin by a carboxylation of Ru-PP with formation (C5 + C = 2C3)
of two molecules of phosphoglyceric acid (PGA). Starting with PGA, all

358 UNITY AND DIVERSITY IN BIOCHEMISTRY

the separate operations of the pentose cycle, and, according to Calvin
(Fig. 94), of the associated photosynthetic cycle, have been reproduced in
vitro using purified enzymes.

Aldolase catalyses the formation of heptulose diphosphate from a
molecule of tetrose phosphate and a molecule of triose phosphate
(C4 + C3 = C7). Phosphatase converts the heptulose-PP to heptulose-P
which, with a further molecule of triose-P, in the presence of transketolase,
forms a molecule of ribose-P and a molecule of ribulose-P (C7 + C3 = 2C5).
The isomerase for pentose phosphates converts this mixture to ribulose-P
which, in the presence of ATP and phosphopentokinase, gives ribulose-PP.

However it still remains, in this enzymatic scheme, to explain the
fixation of CO2 when entering this series of reactions, Calvin has isolated
from spinach leaves and from ultrasonic macerates of the green alga
Chlorella a soluble enzyme which, in the presence of bicarbonate, converts
Ru-PP to two molecules of PGA. He has called it carboxydismutase, because
the carboxylation depends on the oxidation of C-3 of ribulose to a carboxyl
group.

The fact that all the necessary enzymatic tools have been isolated does
not mean that the cycle actually does function. In fact for the reduction of
PGA to a triose, a molecule of ATP and a molecule of TPNH are required.
When we get to Ru-P, a further molecule of ATP is necessary for the
formation of Ru-PP.

The cycle has been completely reconstructed in vitro by Racker (1955),
who has thus achieved the reductive synthesis of a sugar from CO2 and
plant enzyme extracts. The sequence of reactions is as follows:

(1) 3 pentose-P + 3 ATP > 3 Ru-PP + 3 ADP

(2) 3 Ru-PP + 3 CO2 + 3 H2O > 6 PGA

(3) 6 PGA + 6 ATP > 6 DPGA + 6 ADP

(4) 6 DPGA + 6 DPNH + 6 H+ > 6 triose-P + 6 DPN + 6P

(5) 4 triose-P > 2 HPP

(6) 2 HDP + 2 H2O > 2 HMP + 2 P

(7) 1 HMP + 1 triose-P > 1 pentose-P + 1 tetrose-P

(8) 1 HMP + 1 tetrose-P > 1 heptulose-P + 1 triose-P

(9) 1 heptulose-P + 1 triose-P > 2 pentose-P

sum (1-9) 3 CO2 + 9 ATP + 5 HoO + 6 DPNH + 6H+

> 1 triose-P + 9 ADP + 6 DPN -f 8 P

(10) 9 H2O + 9 DPN > 9 DPNH + 9 H+ + 9 O

(11) 3 DPNH + 9 ADP + 9P + 3H+ + 30 > 3 DPN +

9 ATP + 12 H2O

sum (1-11) 3 CO2 + 2 H2O + P > 1 triose-P + 6 O

If we ignore equation (10) and (11) for the moment, we see that overall
(reactions 1-9), to introduce a CO2 molecule into a triosephosphate, three

ENTRY INTO THE BIOSPHERE 359

molecules of ATP and four reduction equivalents (4 electrons) are required.
In nature, according to Calvin, they are provided by the conversion of
electromagnetic energy. We require nothing more. The dynamo of photo-
synthesis (see p. 146) is therefore a means of providing ATP and TPNH.
As for the reductive cycle of photosynthesis, according to this scheme it
consists of a multienzyme system of which the hexosemonophosphate
shunt is another variant.

It is only necessary to compare the pentose cycle of photosynthesis
(Fig. 94) with the hexosemonophosphate shunt in Fig. 38 for their relation-
ship to become apparent. Since the two mechanisms are still partly
hypothetical, it is preferable to await further developments in our know-
ledge before attempting to establish a definite relation between respiration
involving the hexosemonophosphate shunt and photosynthesis. The mode
of formation of a triosephosphate molecule from three CO2 molecules is
shown graphically in Fig. 95.

According to the above views, photosynthesis appears as one aspect
of intracellular regulation. A new stationary state is established when
particles containing the electromagnetic dynamo are illuminated and a
high reduction potential is produced due to a high concentration of TPNH,
and there is an accompanying increase in the amount of ATP. In this
manner photosynthesis is the opposite to respiration which continues
again when illumination is stopped. Thioctic acid, inserted by Calvin into
his description of the electromagnetic dynamo, appears to exert an addi-
tional regulatory action. Thioctic acid is one of the coenzymes for the
oxidative decarboxylation of pyruvic acid and it functions thus in the
disulphide form. When the photosynthetic cycle is functioning it is con-
verted to its sulphhydryl form thus blocking the way to the tricarboxylic
acid cycle, to the corresponding respiratory mechanism, and diverting the
flow of carbon to photosynthesis. Inversely, in the dark, the disulphide
form is re-established and the way is again open for the flow of carbon to
the tricarboxylic acid cycle.

Thus from the above view-point photosynthesis can be regarded as
being simply a special form of the priming reactions set out in Fig. 62.
The pentose phosphate cycle is partly reversed and coupled to a reductive
stage catalysed by triosephosphate dehydrogenase, thus replacing the
oxidation catalysed by glucose-6-phosphate dehydrogenase. In the plant
during photosynthesis, the decarbox}4ation of 6-phosphogluconic acid is
replaced by the carboxylation of Ru-PP. However many of the ideas
expounded in this chapter are hypothetical in nature and other theories
of photosynthesis exist. No one theory can explain all the known facts
and we must wait until the future for agreement to be reached upon the
process which is the principal entry of inorganic carbon and energy into
the biosphere.

360 UNITY AND DIVERSITY IN BIOCHEMISTRY

C. Photosynthesis in the Purple, Brown, and Green Bacteria

The Rhodobacteriaceae are an extensive family of bacteria found in soil
and water. Following Van Niel, they can be subdivided into three groups :

(a) Chlorobacteriaceae. Green sulphur bacteria not requiring organic
growth factors.

{h) Thiorhodaceae. Purple sulphur bacteria not requiring organic growth
factors but able to utilize as hydrogen donors various inorganic sulphur
compounds, organic acids, and in certain cases hydrogen itself.

(c) Athiorhodaceae. Non-sulphur bacteria, purple, red or brown in
colour, requiring various growth factors.

{a) Chlorobacteriaceae

These are anaerobic, and according to Van Niel, HgS is the substance
which reduces the oxidizing radical liberated by photolysis.
Sulphur accumulates in the culture in the form of globules.

[b) Thiorhodaceae

They are anaerobes which do not grow except in the presence of HgS
and light. Like the chlorobacteriaceae they use HgS without oxidizing it
since they are anaerobic, and also like them, they are photosynthetic with-
out liberating oxygen. They do not release sulphur, but sulphuric acid.

light

H2S + 2CO2 + 2H2O > H2SO4 + 2(CH20)

In the place of HgS, they can also utilize sulphite or thiosulphate.

light

4Na2S203 + CO2 + 3H2O > 2Na2S406 + (CH2O) + 4NaOH

The mechanism of this curious piece of photosynthesis is unknown.

(c) Athiorhodaceae

These bacteria assimilate COg in the presence of light, but for this they
require certain organic or inorganic compounds. Fatty acids are good
substrates for their growth. A number of experimental facts indicate
that they act as hydrogen donors, but this idea is still under discussion.
The situation is clearer when we consider the utilization of isopropyl
alcohol. Here, there is no doubt that the organic compound acts as a
hydrogen donor to reduce the [OH] radical leaving acetone behind in the
medium.

The overall equation for the reaction is:

light
CO2 + 2CH3CHOH.CH3 > (CH2O) + H2O + 2CH3CO.CH3

ENTRY INTO THE BIOSPHERE

361

(d) The Pigmentary System of the Greefi, Purple, and Brown Bacteria
This is very similar to that in the green algae and green plants. The
brown or purple colours are due to the presence of various carotenoids.
It is likely that as in diatoms and the blue algae, the carotenoids play a part
in the absorption of light, and transfer the energy thus accumulated to
chlorophyll. The chlorophyll in the Thiorhodaceae and the Athiorhodaceae
is bacteriochlorophyll-«, whilst the green bacteria contain a different
chlorophyll.

Fig. 96 (Errera and Laurent) — Nodules on pea roots.

1 . A young plant which has remained stunted in sterilized sand containing neither
combined nitrogen nor thinned with soil on which legumes have been grown; there
are no nodules on the roots.

2. A young plant of the same age which has flourished in sterilized sand not containing
combined nitrogen but thinned with soil in which peas had formerly grown; nodules
are present on the roots.

3. A root bearing nodules.

II. PHOSPHORUS
The incorporation of phosphorus by green plants is the main means of
entry of this element into the biosphere. If we consider a plant, the
surroundings in which the roots live are very complex. Phosphorus is
present in the form of sparingly, soluble calcium, iron and aluminium salts,
as adsorbed anions on the soil particles and as organic compounds derived
from the corpses of plants, animals and micro-organisms (iron, calcium or
aluminium salts of phytin, and of nucleoproteins). In neutral or calcareous
soils, the calcium salts of organic forms of phosphorus are more soluble

362

UNITY AND DIVERSITY IN BIOCHEMISTRY

and more available for bacterial decomposition. The soluble phosphates are
concentrated by the roots and transported by the xylem in the plant to the
active cells.

III. NITROGEN

In general, plants remove nitrates from the soil. Moreover the other
forms of nitrogen in the soil, such as amino nitrogen and ammonia, are
rapidly converted to nitrates by the action of numerous heterotrophic
bacteria and autotrophic micro-organisms which derive their energy from
the conversion of ammonia into nitrite (nitrous bacteria) or nitrite into
nitrate (nitric bacteria). The absorbed nitrate can be accumulated by the
plant, or reduced to nitrite and then to ammonia which the plant uses,
notably for the formation of amino acid amino groups.

N

End

Fig. 97 — (Guilliermond and Mangenot). Structure of a root nodule
in a legume {Vicia faba).

I. A section along the long axis of the nodule. — R: a transverse section through a root;
N : a longitudinal section of the nodule; c.b. : branched bacterial filament having infected
the root.

II. A transverse section of the nodule. End : endoderm of the root (continuing into the
nodule).

In addition, a limited number of organisms are able to utilize atmo-
spheric nitrogen by reducing it to ammonia or amino groups.

The ocean is poor in nitrogen-fixing organisms. It receives its nitrogen
from inflowing rivers. The Mississippi alone pours 360,000 tons of nitrogen
per annum into the sea in the form of nitrates. The nitrogen of the land
is therefore being continuously removed in this way. It is restored by
putrefaction, by the conversion of the nitrogen of the air into nitrates by
electric discharges during storms, and by the action of nitrogen fixing
organisms, free bacteria or bacteria living in symbiosis with plants.

In fact, the fixation of nitrogen is a very common and wide-spread
phenomenon in the biosphere. Still little is known about its mechanism,
but it does not appear to be everywhere the same.

ENTRY INTO THE BIOSPHERE

A. Fixation in the Root Nodules of Legumes

363

It is to Boussingault that we must give credit for having in 1838 estab-
lished that Legumes fix the nitrogen of the air. After a period of great
confusion, in 1885 Boussingault's views were confirmed by Atwater in
America. Towards the middle of the nineteenth century a relationship was
noticed between this phenomenon and the presence in legumes of the root
nodules which had first been described by Malpighi in 1679. The presence
in the nodules of rod-shaped bacteria associated into filaments had already
been described in 1858, but their presence was incorrectly interpreted by
the morphologists for over thirty years. It was not until 1888 that
Beijerinck isolated these bacteria in a pure culture. The parasitic bacteria
on the roots of legumes are very similar and they are collected together in

Fig. 98 (Guilliermond and Mangenot) — Contamination of cells in the region of a root
nodule, by branches of the bacterial filament (c.b.).

Notice the bacteria contained in the mucilaginous mass of the filament whose branches
insure the infestation of each cell; N and n, nucleus and nucleolus of the infected cells.

he genus Rhizohium. These bacteria do not contain cellulase and they
apparently penetrate the plant at the extreme point of the root hairs which
seem to be devoid of cellulose.

Neither the bacteria nor the plant, considered alone, fix nitrogen. In
association, not only do they fix nitrogen but they excrete nitrogen com-
pounds into the soil: aspartic acid, glutamic acid, oximino-succinic acid, etc.

These lead us to postulate the following mechanism:

Na <- NH2OH

+
CO— COOH

I

CH,— COOH

H2O
C = NOH— COOH CHNH2— COOH absorption
I "^ I "*■ "ito

CH2COOH 4(H)CH2— COOH the plant

Oxaloacetic acid oximino-succinic acid aspartic acid

There is no accumulation of hydroxylamine, so that it is probably
rapidly reduced to ammonia when reacting with the ketonic acid. Alterna-
tively, as believed by some workers, ammonia may be the first compound

364 UNITY AND DIVERSITY IN BIOCHEMISTRY

to be formed from the nitrogen of the air. The essential fact is the pro-
vision of oxaloacetic acid by the host plant.

The nodules contain a haemoglobin (leghaemoglobin) of low molecular
weight (17,000), but is role in nitrogen fixation is unknown.

B. Fixation by Autonomous Organisms

In 1883, Berthelot showed that earth became enriched in nitrogen just
by contact with the air, even if it was sterilized ; he concluded that micro-
organisms capable of fixing nitrogen were present in the soil.

(a) Clostridium

The first of these micro-organisms was isolated by Winogradsky in 1895,
it is Clostridium pasteurianum, sometimes called Amylobacter. In this
anaerobe, unlike Rhizohium, fixation is not inhibited in the presence of
carbon dioxide. The ability to fix nitrogen is widespread in the Clostridia
and it has also been found in another anaerobe, Desulphovibrio, which
reduces sulphates.

(b) Myxophycae {Blue-green Algae)

These algae and especially those of the genus Nostoc and the genus
Anabaena, fix nitrogen, but rather slowly. The fixation is inhibited by
ammonia and nitrates and is not quantitatively important in soils.

(c) The Purple Bacteria

Many of the bacteria in the Athiorhodaceae group (non-sulphur purple
bacteria) fiix nitrogen. This fixation is coupled to growth and only occurs
in the presence of light and oxygen.

(d) Azotobacter

These bacteria are exceptionally large and resemble the blue-green
algae. They easily form gums and they are obligatory aerobes. Fixation
of nitrogen only occurs in the presence of a source of carbon and is probably
coupled to oxidation. A part of the nitrogen fixed is excreted into the
medium in the form of amino acids. Again, ammonia itself or a precursor
of it is combined with the dicarboxylic acid. As in the other cases of
fixation, the mechanism of the process is shrouded in mystery.

IV. SULPHUR

Plants absorb and assimilate sulphates. Soil contains a great deal of
calcium sulphate. Bacteria exist which reduce sulphates to HgS. Such
bacteria live in muds, animal intestines, etc., and everywhere where
organic materials are putrefying.

SO4- + 4H2 -^ HaS + 2H2O + 20H-

ENTRY INTO THE BIOSPHERE 365

The sulphides formed in the soil or in water can be again converted into
sulphates, either by a purely chemical action or by the action of various
bacteria, colourless sulphur bacteria, purple sulphur bacteria, etc.

REFERENCES

Hill, R. and Whiltingham, C. P. (1955). Photosynthesis. Methuen, London.
Lees, H. (1955). Biochemistry of Autotrophic Bacteria. Butterworth, London.
Bassham, J. A. and Calvin, M. (1957). The Path of Carbon in Photosynthesis
Prentice-Hall, Englewood Cliffs, N.J.

CHAPTER II

DEPARTURE FROM THE BIOSPHERE

The earth and the surface waters are the natural tombs of plants and
animals. In these regions of the lithosphere and hydrosphere, the materials
of the biosphere return to the inorganic world : the nitrogen of proteins
becomes ammonia and nitrate, carbon is oxidized to carbonates and the
other elements return to their inorganic forms. Elsewhere, too, living
organisms are returning these elements by a continual rendering of respira-
tory carbon dioxide and metabolic excreta. The excreta and corpses are
mineralized in the soil and in water by the action of micro-organisms.
The latter, likewise, autolyse when unfavourable conditions interrupt their
multiplication.

Particularly important are the processes by which nitrogen and carbon
leave the biosphere to re-enter the inorganic world.

I. AMMONIFICATION IN THE SOIL

The dead bodies and excreta of living beings are attacked in the ground
by the exoenzymes of many bacteria. For example, the exoenzymes of
many Clostridia attack this dead matter and the proteins are converted to
amino acids. Many bacteria release ammonia from these amino acids.
The most active ammonifying organisms are Bacillus mycoides, Proteus
vulgaris and various actinomycetes. Quantitatively the most important
process is oxidative deamination (p. 210).

The bacteria of the soil can also accomplish a deamination by the
removal of ammonia to produce a double bond

R_CH2— CHNH2— COOH -^ R— CH = CH— COOH -f NH3

In this way bacteria of the coK-typhosum group can convert histidine to
urocanic acid.

CH==CH— CH2CHNH2COOH CH=:CH— CH=CHCOOH

I I

HN N -^ HN N -t-NHs

\ / \ //

C C

H H

Histidine Urocanic acid

366

DEPARTURE FROM THE BIOSPHERE 367

In the same way aspartic acid is converted to fumaric acid by E. colt.

CHNH2— COOH CH— COOH

I ^ II +NH3

CH2— COOH CH— COOH

The removal of HgS from cysteine is followed by the same process,
which can be accomplished, in part, by Proteus vulgaris.

CH2SH CH2 CH3 CH3

1 — H2S II I H2O I

CHNH2 -> CNH2 -» Cr^NH -> C=0 H- NH3

COOH COOH COOH COOH

Another type of deamination is reductive deamination as carried out
anaerobically by the Clostridia. Glycine gives acetic acid, alanine and
serine give propionic acid, etc. Although evidently hydrogen plays some
part in the reaction, only amino acids are necessary. For example, two
moles of glycine are reductively deaminated in the presence of CI. sporo-
genes whilst at the same time a mole of alanine is oxidized to acetic acid.

CHa— CHNH2— COOH + H2O -^CHa— CO— COOH + NH3 + 2 (H)
CH3— CO— COOH + H2O -> CH3— COOH + CO2 + 2 (H)
The hydrogen is then accepted by the glycine

2 CH2NH2— COOH + 4 (H) -^ 2 CH3— COOH + 2 NH3

The amino group of amino acids is never hydrolysed directly by bacteria,
but the latter can liberate NH3 by hydrolysis of amides such as asparagine
and glutamine.

R— CONH2 + H2O -> R— COOH + NH3

A source of ammonia in the soil is the urea arising from the hydrolysis
of arginine by various micro-organisms to ornithine and urea (micrococci,
Bacillus subtilis). Certain bacteria (Streptococcus fecalis, Streptococcus
hemolyticus, Micrococcus aureus; these organisms are parasites and play
no part in the soil economy) contain an arginine-hydrolase and can
perform a double hydrolysis of arginine to produce ornithine, two molecules
of ammonia, and COg.

Certain other bacteria can form citrulline by a hydrolytic deamination
of the =NH of arginine, but the citrulline formed is only metabolized
very slowly.

Because they contain urease in their cells, many bacteria can hydrolyse
urea, either derived as outlined above, or produced as an animal excretory
product.

368

UNITY AND DIVERSITY IN BIOCHEMISTRY

NH2— CO— NH2 + 2H2O ^ (NH4) 2— CO3 -> 2NH3 + CO2

If these above reactions are the source of ammonia in the soil, the
degradation of amino acids and similar substances is complicated by the
occurrence of numerous side-reactions which can inhibit ammonia produc-
tion and consequently the nitrogen cycle.

C^15

,te

U*

Citrulline + NH,

pco'

ilv^

S^v.

Arginine

hemolyticus
aureus

>- Ornithine + 2 NH3 + COj

o

Ornithine + urea

Examples are : the uptake of amino acids resulting from the hydrolysis
of dead bodies by the protoplasm of bacteria, or by fungi ; and in the soil,
the formation of nitrogen-containing humus, by combination of carbo-
hydrates and nitrogenous compounds.

But the most important side-reaction is bacterial decarboxylation,
brought about by organisms of the coli group, by Clostridia, etc. and by
fungi.

R— CH— COOH > R— CH2NH2 + CO2

NH2

The amines formed are basic, toxic to animals, and resistant to bacterial
decomposition. Their oxidation only occurs in an alkaline medium and
they are formed in an acid medium, so that the bacteria which produce
them do not oxidize them.

II. THE EXIT OF CARBON FROM THE BIOSPHERE
Carbon disappears from the biosphere chiefly as CO2 formed in the
tricarboxylic acid cycle and eliminated by the organism. Equilibrium
between this mechanism of loss and entry of carbon due to photosynthesis
would not be maintained if there were not other ways in which CO2 is lost.
The bacterial mineralization of corpses is partly responsible for filling
the gap.

DEPARTURE FROM THE BIOSPHERE 369

A. The Loss of Carbon present in Carbohydrate Macromolecules

(a) Starch

Bacteria and fungi are able to secrete a amylases into the surrounding
medium and in particular this can be done by Bac. subtilis, mesentericus,
macerans and polymyxa.

(h) Pectins

As we have already seen these are methylated polymers of D-galacturonic
acid (polyuronides and not polysaccharides).

The chief organisms attacking pectin are found among the Entero-
bacteriaceae and such spore bearers as Bac. macerans and most of the
Clostridia.

The breakdown of pectin is a complex process and takes place in several
stages.

1. Conversion of protopectin to pectin (this occurs during the retting
of flax). The enzyme responsible is protopectinase, which is present in
Aspergillus, CI. felsineum, etc.

2. Demethylation of pectin to pectin acid. The enzyme is pectase which
is found in many bacteria.

3. Hydrolysis of the 1-4 linkage, spHtting the macromolecule. Here the
corresponding enzyme is pectinase which is present in Asp. oryzae,
Rhizopiis tritici, etc.

(c) Cellulose

Since cellulose constitutes the major part of the insoluble material in
plants, the speed at which it is broken down is one of the important factors
in the carbon cycle.

A number of micro-organisms capable of decomposing cellulose exist :
myxobacteria, Clostridia, Actinomycetes and many fungi. The presence
of a soluble cellulose has only been shown in a few cases. In other cases,
the way in which the cellulose is broken down has not been decided.

B. The Loss of Carbon present in Amino Acids

The ketonic acids which result from the action of microbial proteases
and the oxidative deamination of the resulting amino acids, can be directly
reduced to hydroxy-acids

2H

R— CO— COOH > R— CHOH— COOH

The ketonic acids can also be decarboxylated with the loss of one carbon
atom to form an aldehyde.

R— CO— COOH -> COo + R— CHO

370 UNITY AND DIVERSITY IN BIOCHEMISTRY

The aldehyde may be dehydrogenated to an acid having one carbon
atom less than the starting amino acid, or the aldehyde may be reduced to
an alcohol

R— CHO + 2H ^ R— CH2OH

However most of the ketonic acids are oxidized by micro-organisms and
CO2 is formed. Soil consists of particles separated by water or a gaseous
atmosphere. This atmosphere contains more COg than the air. When the
soil is poorly aerated and there is much organic matter present, bacterial
action produces not only COg, but also some methane. The soil of rice-
fields contains practically no oxygen but there is much hydrogen and
methane present.

REFERENCE
Thimann, K. V. (1955). The Life of Bacteria. Macmillan, New York.

CHAPTER III

THE CYCLES

I. THE CARBON CYCLE

During photosynthesis, COg is removed from the atmosphere and from
the hydrosphere. A part of this COg is returned almost immediately during
the respiration of plants. The remainder is, for the most part, returned
indirectly by the respiration of animals and during the putrefaction of
dead plants and animals. This is known as the biological carbon cycle.

The CO2 content of the atmosphere hardly varies and this is due to the
buffering of drastic fluctuations by the CaC03-Ca(HC03)2-COa
system of the ocean. The main path of the carbon cycle is through the
biosphere and the ocean. Carbonates are being continually diverted in the
form of sediments, and organic carbon is being held up during its series of
transformations in the form of fossilized carbon.

It is possible to calculate that since the start of the laying down of these
two types of sediment, twelve times the total amount of COg in the atmo-
sphere has been trapped in the form of sediments. This removal of carbon
has taken place gradually and has been compensated by CO2 of volcanic
origin.

The autoregulation of the carbon cycle, which maintains the COo of the
atmosphere at a constant level, depends on the one hand on the COg—
bicarbonate-carbonate system of the air, seas and sediments, and on the
other hand it depends on photosynthesis, the intensity of which is regulated
by the concentration of available COo.

The carbon cycle is not at all dependent on the presence of animals or
plants. It could continue in the presence of micro-organisms alone and
with special intensity in the oceans where the photosynthesis is eight
times more intense than that due to land plants. This marine photo-
synthesis is due to the presence of Diatoms and the Dinoflagellates of
phytoplankton. Figure 100 shows a carbon cycle which is entirely microbial
in character.

II. THE NITROGEN CYCLE

The main stages of this cycle are the fixation of nitrogen or nitrates by
plants and certain bacteria, the mineralization of proteins to form ammonia
and the conversion of this ammonia into nitrates. Fig. 101 shows the various
stages of the cycle.

371

372

UNITY AND DIVERSITY IN BIOCHEMISTRY

u

a

O

<u
J3

.a

•a
•S

S

0)

u

a

u
a

c

o

S

3
O

%\

1

v\

1

M

V\

u

'^o \

o

% \

ca

^\^

C

\>^

^_w. "^

<C ff. i UOUBJfdlODJ J

o B>

U c

o

2 to

'l-i V

Oh j:

^L

c
o

09

c

d

CO

THE CYCLES

373

C02

Air, and dissolved air

Respiration
Decomposition

Organic matter of algae and
photoautotrophes in general
(chiefly in water)

Organic matter of autrotrophic

bacteria (chimioautotrophes)

(chiefly in soil)

Organic matter of
saprophytic micro-organisms

Fig. 100 (after Butlin andj^Postgate) — A microbiological carbon cycle.

374

UNITY AND DIVERSITY IN BIOCHEMISTRY

O

Nitrobacter

Various organisms g

u

a

CO

O

o

C
<u

60
O

X,

c
c

w

S

o

THE CYCLES

375

Atmospheric nitrogen

Nitrosomonas

Ammonia

CI. zvelchii
Desulpkovibrio, etc.

"Nitrite

Bacterial proteins
(autotrophes and heterotrophes)

Nitrate

Fig. 102 — Microbial nitrogen cycle.

Bacterial oxidation

Sulphur

HgS

a

Bacterial oxidation

Bacterial desulphuration

Sulphates

*o. /\ S of plant organic compounds

Excreta

S of animal organic compounds

Urine

Fig. 103— The sulphur cycle.

376

UNITY AND DIVERSITY IN BIOCHEMISTRY

If we have a heterogeneous microbial population, the cycle can operate
without the intervention of animals, and even without that of plants, as
Fig. 102 demonstrates.

III. THE SULPHUR CYCLE

This is somewhat analogous to the nitrogen cycle. Like nitrates, sulphates
are utiHzed and reduced by plants. In its reduced form ( — SH groups),
like nitrogen in its reduced form as the amino groups of amino acids,
sulphur plays a part in the construction of vegetable proteins, from whence
it enters the animal organism. A part of this sulphur appears in the
oxidized form in the urine (derived from sulphuric acid). When the plant

Sulphur

Sulphides

Beggiatoa
Thiobacillus spp.^
Chromatium ,
ChlorobiuTTi/

Beggiatoa

^Thiobacillus spp.
, Chromatium
Chlorobium

Thiobacillus spp.

Desulfovibrio
Fig. 104 (Butlin and Postgate) — A microbial sulphur cycle.

Sulphates

or animal putrefies, the sulphur is liberated by many aerobic and
anaerobic bacteria as HgS (analogous to deamination with the production
of NHg). Part of naturally occurring RgS also comes from the removal of
sulphur from sulphates by bacterial processes.

The opposite of the reduction of sulphates is a process corresponding to
nitrification, by which bacteria convert HgS and elementary sulphur to
H2SO4. Other bacteria can reduce elementary sulphur to HgS.

As in the case of the nitrogen cj^cle, animals and plants are not indis-
pensable to the operation of the sulphur cycle. In nature one sometimes
encounters this cycle maintained by a microbiological association of auto-
trophic bacteria, algae and protozoa, the name sulphuretum is applied to
this community which can be found in clays, muds and in stagnant waters.
Figure 104 shows a bacterial sulphur cycle.

THE CYCLES 377

IV. THE CIRCULATION OF PHOSPHORUS

The circulation of this element so necessary to life takes place exclusively
by way of compounds in which the phosphorus is in its highest state of
oxidation and hydration. Orthophosphoric acid, originating in the in-
organic world, during its passage through the living world undergoes no
chemical change apart from the formation of salts and esters (calcium
phosphate in bones, phosphatides, nucleoproteins, sugar phosphates, etc.).

Before entering the living world, the phosphoric acid in inorganic com-
pounds must first be mobilized. This is brought about by the liberation
of various strong acids into the soil by numerous aerobic and anaerobic
micro-organisms. The COg liberated by the roots of plants and by
bacteria also plays a part. Once rendered soluble the phosphates may be
reprecipitated or assimilated by plants and then passed to animals in
organic or inorganic combination. The excreta of animals and the dead
bodies of plants and animals return to the soil the phosphates which have
been removed. In this way a local accumulation of phosphate may be
obtained and used as fertilizer.

One remarkable aspect of phosphorus metabolism in the biosphere is
the increase in phosphate concentration with depth in the ocean. It
appears that the phosphorus gradually passes into the sediment at the
bottom and it can be calculated that several times the amount present in
the ocean has been removed in this way and been replaced by phosphorus
from the lithosphere brought down by the rivers. Compared to the removal
of this element the return of phosphorus to the continents is ver}^ much in
arrears since it depends on the uptake of food from the sea by animals,
especially man and birds. The fishing industries of the world take around
60,000 tons of phosphorus (25 to 30 million tons of fish). Sea birds bring
back to the land around ten times this figure.

Thus there is a considerable deficit between the 137 x 10^ tons of
phosphorus added to the ocean each year from the continents and the
amount returned to the land by the feeding and excretion of marine
animals. As a result there is a constant accumulation of phosphorus in the
depths of the oceans and it can no longer be used to support terrestrial life.

V. THE METABOLISM OF THE BIOSPHERE

The cycles we have just described show, for the principal elements con-
cerned, the entry of these elements into the biosphere from the inorganic
world and their return to the inorganic world. Taken together these cycles
describe the metabolism of the biosphere and they regulate its mass and
distribution. Each region of the biosphere, made up of a community of
organisms and their surroundings, is a functional unit and is termed an
ecosystem.

378 UNITY AND DIVERSITY IN BIOCHEMISTRY

From the biochemical point of view the organisms which make up an
ecosystem are of four types : producers, consumers, those which bring
about decomposition, and those which accompHsh transformations of
material. In the terrestrial region, for example in soil, the producers are
mainly green plants although autotrophic bacteria also play a part. All the
consumers are heterotrophes, i.e. herbivores consuming green plants
(insects, rodents, ruminants, etc.), primary carnivores who consume the
herbivores, and secondary carnivores who feed on primary carnivores, etc.
Parasites are also consumers. The decomposing organisms are those that
attack the dead bodies of producers and consumers and turn their sub-
stance into the inorganic state of COg, ammonia, HgS, etc. This is accom-
plished by bacteria and fungi.

The transforming organisms alter the inorganic compounds resulting
from the activities of the above and convert them to compounds which
can be used by the producers ; nitrates and sulphates for example.

It appears therefore that an ecosystem can exist very well if it is made
up of producers and decomposing and transforming organisms. The
consumers, including man, are only an unessential intermediate in the
metabolism of the ecosystem.

The components of marine ecosystems differ from those of terrestrial
ecosystems. In the open sea for example, the principal producers are the
plankton, especially diatoms, which form what has been called "the ocean
pasture". The amount of growth depends on seasonal conditions and
photosynthesis only occurs where light is able to penetrate. Also, in
addition to COg, nitrates, sulphates and phosphates are required. In
winter when the ocean waters become agitated and are mixed, these salts
are brought to the surface and rapid growth can begin as soon as there is a
sufficient number of hours of sunlight. But gradually the inorganic salts
are used up and the herbivorous consumers, especially the copepods,
graze the ocean pasture and devour it. During the summer, owing to
thermal stratification of the water and because the dead organisms sink into
the depths, the situation remains stationary. When autumn comes, the
surface temperature falls and agitation of the waters increases ; the upper
layers are resupplied with inorganic nutrients and a new growth of phyto-
plankton begins which is soon arrested by being consumed and by the fall
in temperature and the amount of light. Winter brings about the great
mixing of waters which, in the sea, corresponds to the tilling of the soil.

We have already listed the copepods among the marine herbivorous
consumers. They are not the only examples, many molluscs and even
certain fish belong to this group. But herbivorous consumers are less
common than on land and they are almost always small in size (copepods,
etc.). These small herbivores are eaten by numerous marine animals and
notably by the whale. However most marine carnivores are dependent on

THE CYCLES 379

the consumption of larger animals. The decomposing and transforming
organisms in the marine habitat are, as on the land, micro-organisms.

Considered as a whole, the biosphere is nourished essentially by COg,
nitrogen, water, sulphates, nitrates and phosphates. The greater part of
the energy entering it is of solar origin.

One important aspect of the metabolism of the biosphere is the topo-
graphy of its distribution. On land, the products of the activity of degrad-
ing organisms may be retained in the soil, although there is always a loss
which may be considerable in acid soils or where there is excessive rain.
In the sea, dead bodies end by sinking into the depths and they may be
displaced by the currents so that the products of their transformation may
be situated far away from the place where the macromolecules were
synthesized. In the depths of the ocean phosphates and nitrates may be
deposited far away from the places where photosynthesis is occurring
but they may be brought to the surface again by vertical currents. Although
during the year the displacement downwards and the displacement upwards
may approximately cancel each other out, mineral salts are being con-
tinuously poured into the sea and a considerable amount is accumulated
on the sea bottom and is inexorably removed from the terrestrial habitat.

On the other hand, the return to the inorganic form of what has been
assimilated by the biosphere may be blocked by hold-ups in the mineral-
ization process. The humus in the soil is one of these hold-ups and it is
beneficial in its influence on the physical properties of the soil. But peat,
lignite and coal show how there has been a long hold-up in the carbon cycle
in certain areas. Chalk, coral, etc., are further examples of stagnation in the
same cycle. Another example is the accumulation of guano in certain islands
in Peru. No doubt the nitrogen of the atm^osphere is an inexhaustible
source, but the COg of the air is not. Even in the equilibrium state of
the biosphere at the present time, the low level of COg in the atmosphere
under bright conditions limits the intensity of photosynthesis which would
be much greater if more COg was available.

The low concentration of COg in the atmosphere is the result of an
equilibrium between producers and consumers. If this small amount was
no longer present, life would stop.

As for phosphorus, it can only be Hberated slowly from rocks and marine
deposits. The most rapidly available source is provided by the decomposi-
tion of organisms. This is the vulnerable point in the metabolism of the
biosphere. The increasing removal of phosphorus from the land to
the sea is a menace to all terrestrial life, and it may make the colonization
of the continents a mere episode between two eras of marine existence,
unless the return of phosphorus to the land is increased by man, perhaps
by the realization of his old dream of utilizing the vast resources of the
oceans.

380 UNITY AND DIVERSITY IN BIOCHEMISTRY

REFERENCES

Aller, W. C, Emerson, A. E., Park, O., Park. T and Schmidt, K. P., (1949).

Principles of Animal Ecology. Saunders, Philadelphia.
Clarke, G. L. (1954). Elements of Ecology. Wiley, New York.

Index

Acceptors 144, 153, 159, 161, 162, 174,

193, 207, 211, 234, 285
Acetabidaria 266
Acetaldehyde 141, 163, 188, 191, 194,

226, 254
Acetalphosphatides, see Plasmologens
Acetate, see Acetic acid
Acetic acid 8, 91, 221, 235, 236, 237,

251, 268, 367
Acetoacetic acid 2, 14, 17, 217, 219,

224, 235, 237
Acetoacetyl-CoA 197, 205, 226, 235
Acetone 229, 230, 268
AT-Acetyl a-aminoadipic-e-semialde-

hyde 218
Acetyl group, donor 72

transfer 175
Acetylation, amine 175
Acetylcholine 161
AT-Acetylchondrosamine 93
Acetyl-CoA 72, 144, 195, 196, 197,
199, 203, 217, 224, 225, 229, 235,
289, 308
Acetylgluocosamine 69
Acetylhexosamine 92
AT-a-Acetylornithine 245
Acetylose 85

Acetylphosphatase 156, 230
Acetyl-phosphate 229
Acids, aliphatic 8-14

amino, see Amino acids

deoxyribonucleic, see Deoxyribo-
nucleic acids

dibasic 11, 230

fatty, see Fatty acids

hydroxy 11,12,213,370

imino 29, 212, 215, 224

keto- 13, 14

polybasic 1 1

ribonucleic, see Ribonucleic acids
Aconitase 165, 202, 204, 231
cis-Acomt\c acid 203
Acrolein 9
Acrylic acid 9

Activation 151, 161, 176, 234, 261,

262, 263
Activators 152
Active acetate, see Acetyl-CoA
Adenine 53, 68, 70, 107
Adenosine 59, 67

diphosphate (ADP) 67, 68, 143,
147, 160, 180, 189, 191, 195, 206,
227, 259, 285, 358
monophosphate (AMP) 67, 235,

256, 260, 285
triphosphate (ATP) 68, 142, 143,
146, 160, 175, 188, 191, 195, 206,

223, 225, 227, 232, 252, 256, 280,
309, 355, 357, 360

S-Adenosylhomocysteine 233
S-Adenosylmethionine 233, 234, 243
ADP. See Adenosine diphosphate
Adrenal cortex, cells 315
Adrenaline 284
Aerobacter aerogenes 248
Aetiocholane 42
Agmatine 210
Alanine 26, 80, 166, 210, 220, 223,

224, 228, 241, 247, 267, 367
racemase 1 66

^-Alanine 25

Alanylalanine 77

Albumins 97, 101

Alcohol dehydrogenase 162, 195

Alcoholic fermentation, see Fermenta-
tion

Aldehyde, acceptor, see Phospho-
glyceraldehyde
oxidase 71, 164

Aldohexoses 17

Aldolase 103, 165, 358

Aldopentoses 1 7

Aldoses 12, 15, 16

Alg« 291, 325, 326, 327, 354, 364

Alginic acid 90

Allantoic acid 342

Allantoine 342

Allocholestane 40

381

382

UNITY AND DIVERSITY IN BIOCHEMISTRY

AUohydroxy-L-proline 80
D-Allose 16
L-AUose 16
D-Allothreonine 30
L-AUothreonine 30
Alloxazine derivatives 54—55
Allyl alcohol 9
D-Altrose 16
L-Altrose 1 6
Amanita phalloides 80
Amide link 276
Amine acetylation 175
D- Amino acid oxidases 210, 280
L-Amino acid oxidases 210, 214
Amino acids 7, 24-30, 100, 226, 267,
294, 312, 337, 368, 369

activated 263

aromatic 248, 249

deamination 210-212

decarboxylation 209-212

degradation 209, 215-223, 224,
228

free, in proteins 102

intracellular pool 264

oxidation 223, 224

precipitation 103

sequence determination 104, 105

short chain 242

titration 100

transamination 213—214

transdeamination 2 1 4—2 1 5
a-Aminoacrylic acid 212
a-Aminoadipic acid 218, 251, 252
/)-Aminobenzoic acid 248
a-Aminobutyric acid 25, 210, 245,

247, 268
Aminoethanol 74

4-Aminoimidazole-5 -carboxamide 240
Aminoimidazoleribotide 256
8- Aminolevulinic acid 238, 239, 243
Aminopeptidases 154
Ammonia, 167, 181, 215, 362, 363
Ammonification, soil 366-368
AMP. See Adenosine monophosphate
Amphibia 318, 329, 330, 344
Amylase 58, 154
a-Amylase 155,369
]8- Amylase 88, 154
Amylobacter 364
Amylo-l,6-glucosidase 262
Amylo-transglucosidase 158
Amylo-(l ,4^ 1 ,6)-transglucosidase 262

Amylopectin 88, 89, 263

Amylose 87, 88, 158, 161, 263

Analogy 334-336

Animals, see Invertebrates, Reptiles,

Vertebrates, etc.
Annelids 117, 294, 334, 336
Anserine 78, 79, 232
Antheroxanthin 35
Anthocyanidins 50
Anthocyanins 50, 305
Anthoxanthins 48, 305
Antibiotics 79
Antibodies 301
Antigens 92, 302
Aphanine 34
Aphrodite aculeata 295
Apoenzyme, 1 74
Arabinose 91
D-Arabinose 16, 22
L-Arabinose 17, 22, 254
Area 295

Arenicola 294, 300, 301, 335
Arginase 158
Arginine 26, 94, 96, 99, 101, 158, 209,

243, 244, 245, 268, 294, 310, 368
phosphokinase 160
L- Arginine 210
Arginosuccinic enzyme 310
Argon 2, 3
Argon-40 3
Arthropods 330, 331
Asparaginase 157
Asparagine 99, 102, 158, 214, 245, 246,

366
Aspartase 245
Aspartic acid 101, 157, 207, 210, 215,

221, 224, 245, 246, 251, 268, 310,

363, 366
j8-Aspartylphosphate 246
Aspergillus oryzce 369
Astacine 36
Astaxanthin 36
Athiorhodaceas 360
Atmosphere 1, 3, 4, 379
Atoms, nucleus 183
number 183
radioactive 183
ATP. See Adenosine triphosphate
Auroxanthin 35
Autotrophs 179, 184, 352, 353
Autotrophy 353
Autoxidation 351

INDEX

383

Azafrine 37
Azotobacter 364

lactis aerogenes 265

macerans 369

viesentericus 369

mycoides 366

prodigiosus 366

subtilis 367, 369

Bacteria 229, 251

ammonification 366—368

autotrophic 351—353

mineralization 368-370

propionic acid 231

purple, brown, green 360-361, 364
Bacteriochlorophyll 122
Baikiaine 29
Barbituric acid 52
Benzaldehyde 14
Benzene ring, biosynthesis 248
Benzopyrilium 50
Benzoquinone 357
Betabacterium vermiforme 89
Betaine 232, 233
Bile acids 40, 42, 237, 310
Bilirubin 311
Biochemical parallelism 335
Bioluminescence 145
Bioses 14
Biosphere 1-8, 179, 374

metabolism 349-365
Biosyntheses 229-269
Birds 290, 320, 339
Blood, see also Erythrocytes, Haemo-
globin, Leucocytes, etc. 292, 293,
334, 336
Blood group polysaccharides 92
Blood plasma proteins 126
Blood platelets 314
Bonds, see also Linkage 10, 57-83, 99,
114, 117

acyl phosphate 139, 142

amide 77, 260, 276

amidine 158

anhydride 61, 67

C-C 164, 230

C-N 164

C-S 164

covalent 57, 83, 84, 112, 117

energy liberation 139-144

energy-rich 72, 137, 138, 142, 180,
190, 199, 205, 223, 225, 228, 280, 351

enolphosphate 139

ester 61-77, 139, 276

guanidine phosphate 139

hydrogen, see Hydrogen bonds

internucleotide 108

ion-dipole 113

oside 57-61, 85, 139, 254

~P 144

peptide 77-83, 139, 154, 260, 263,
266, 275

pyrophosphate 139, 142, 146, 179,
262, 263

thioester 139, 143

thiophosphate 139
Bone 312
Butanoic acid 9
Butyric acid 9,12,223
Butyryl-CoA 197

Cadaverine 210
Calcium 5
Camposterol 42
Capsanthin 36, 37
Capsorubin 36, 37
Carbamylaspartic acid 257
Carbamylglutamic acid 309, 311
Carbamylphosphate 255, 257, 258
Carbohydrases 152, 154
Carbohydrates 351, 352

catabolism 194

metabolism 188, 308
Carbon, Ci units 229

C2 fragments 235

C5 branched chain 237

cycle 371-373, 379

energy 351-361

isotopes 183
Carbon dioxide 2, 3, 356, 357, 360,
371, 372, 379

exit from biosphere 368-370

fixation 230, 231
Carbosynthetases, see Carboxylases
Carboxydismutase 358, 359
Carboxyhasmoglobin 117, 294
Carboxyl group, activated 261, 263,

266
Carboxylases 66, 164, 195, 212
Carboxylations 230—232
a-Carboxylation 230, 232
j8-Carboxylation 230-232
Carboxypeptidases 1 54
Camosine 78, 79

384

UNITY AND DIVERSITY IN BIOCHEMISTRY

Carotene, trans-S and poly-cis 304

a-Carotene 32

j6-Carotene 32

^-Carotene, aW-trans 304

y-Carotene 32

a-Carotenepoxide 33

Carotenes 32, 37, 325

Carotenoids 31-38, 235, 267, 289, 290,

304, 354, 361
Cartilage 313
Caryolymph 279
Casein 264

Catalases 46, 120, 121, 212, 268
Catalysts 152
Cathespin 154, 279
Cellubiase, see ^-Glucosidase
Cellobiose 58, 85, 86
Cellophane 87
Cells, differentiation 305-316

nucleus 274, 279-280

nutrients 179

regulation 282—286

topochemistry 273-280

wall constituents, algae 326

work 145, 146
Cellulases 155, 370
Cellulose 59, 85-87, 154
Cephalins 74, 252
Cerebrosides 12, 76
Ceriodaphnia sp. 300
Chelydra serpentina 318, 321
Chimiosynthesis 351
Chironomus sp. 295, 300, 301
Chitin 22, 85, 91
Chitinase 90
Chitobiose 91
Chlorella 359, 367
Chlorine 5

Chlorobacteriaceae 360
Chlorobhim 376
Chlorocruorin 117, 118, 120, 333, 334,

335, 336
Chlorocruoroheem 117
Chlorocruoroporphyrin 117, 120
Chlorophyllase 122
Chlorophyll-proteins 121
Chlorophylls 30, 121-123, 291, 325,

354, 355, 361
Chloroplasts 354
Cholestane 40

Cholesterol 7, 39, 41, 127, 236, 237,
239, 337

Cholic acid 42

Choline 74, 232, 234, 252, 253

acetylase 161

dehydrogenase 164

phosphokinase 252, 253
Chondroitin sulphate 93
Chroman 47, 48
Chromatin 280
Chromatium 211
Chromonemata 279
Chromosomes 111
Chrysanthemaxanthin 35
Chymotrysinogen 103
^-Citraurine 37
Citric acid 12, 51, 165, 202, 205, 223,

228
Citric dehydrogenase 202
Citroxanthin 32
Citrulline 25, 244, 245, 309, 310, 311,

368
Classification 323, 334
Clionasterol 42
Clostridia 364, 367, 368, 369
Clostridium felsineum 369

kliiyveri 1 98

pasteurianum 369

Sporogenes 367

welchii 374
Clupeine 96
Cocarboxylase 67
Coenzyme I, see Diphosphopyridine

nucleotide
Coenzyme A 67,72,83,161,175,196,
199, 204, 207, 217, 219, 253, 259,
260
CoA transferases 162
Codehydrogenase, see Diphosphopyri-
dine nucleotide
Coenzymes 47, 55, 67, 152, 171, 173-

175, 188, 284
Cofactors 152
Collagen 313
Colloids 84
Colnbridce sp. 341
Complexes 113, 114, 122, 123
Condensing enzyme 204
Copper, protein bound 123
Coproporphyrins 44, 45
Coprostane 40

Cori ester, see GIucose-1 -phosphate
Corticosteroids 237
Corynebacterium diphtherice 92

INDEX

385

Covalency, see Bonds, covalent
Cozymase, see Diphosphopyridine

nucleotide
Creatine 159, 233

phosphokinese 160
Crocetin 37
Crotonyl-CoA 197
Cryptoxanthin 34, 37
Cuproproteins 1 24
Cyanocobalamin 72, 73, 235
Cyclopentane 38
Cyclostomes 328, 329
Cystathione 243, 246
Cysteamine 72
L-Cysteic acid 210
Cysteine 26, 78, 100, 101, 221, 223,
242, 243, 245, 246, 268, 366

degradation 222, 224
Cysteine-sulphinic acid 222
Cystine 26, 242, 243, 294

degradation 222, 224
Cytidine 242, 255

monophosphate 257, 258

-triphosphate 253
Cytidinediphosphatecholine 252, 253
Cytochrome 199

a 120, 141, 206, 208

fli 120

a. 120

03 120, 163, 164, 206, 208, 278

b 120, 206, 208

c 119,141,206,208,209,295
Cytochrome oxidase, see Cytochrome a^
Cytochrome reductase 208
Cytochromes 119-120,208,353
Cytoplasm 274-277, 284, 313
Cytosine 54, 107, 111, 255

Daphnia 300, 315, 316
Deamidases 153, 156
Deamidations 212-213
Deamidinases 153, 158
Deaminases 153, 156
Deaminations 210-212, 366-368

oxidative 210

non-oxidative 212
Decanoic acid 9
Decarboxylases 165, 210-213
j8-Decarboxylases, oxidative 213
Decarboxylation, amino acid 209, 210

bacterial 368

Ketonic acids, 165, 212, 213

oxidative 204-206
Dehydrogenase AH, 198, 201
Dehydrogenation 207
5-Dchydroshikimic acid 248, 249
5-Dehydroquinic acid 248, 249
Denaturation 93, 98, 100
Deoxypentoses 268
Deoxyribonucleic acid (DNA) 107,

109, 110, 111, 267, 279
Deoxyribose 23, 59, 254
-1-P 65
-5-P 253
2-Deoxy-D-ribose 107
Desoses 22

Desiilfovibrio 364, 373, 376
Desulphydrase 222
Detoxication 310
Deuterium 183
Deuteroporphyrin 45, 46
Dextranases 155
Diacylphosphatidic acid 252
Diamagnetism 114
a,a'-Diaminopimelic acid 251, 252
Differentiation, cellular 305-316
Diffusion 146
Digestion 340
Digestive tract, cells 308
1,2-Diglyceride 252, 253
Diholosides, see Disaccharides
Dihydroorotic acid 257
Dihydropyrrol 122
Dihvdroxvacetone 15, 17, 22, 194

-P 166
Dihydroxy-methylvaleric acids 246
5,6-Dimethylbenzimidazole 72
6,7-Dimethyl-9-D-ribityl-isoalloxazine,

see Vitamin Bo
Dimethylglucose 85
Dimethylglycine 234
Diphosphofructosephosphatase 191,

195
1,3-Diphosphoglyceric acid 142, 143,

189, 195, 284, 285
2,3-Diphosphoglyceric acid 166, 175
Diphosphopvridinenucleotide (DPN,

DPN',bPNH) 70,142,161,162,

174, 189, 195, 196, 200, 205, 207,

209, 213, 215, 225, 259, 278, 280,

301, 342, 359
Diphosphothiamine (DPT) 67, 190,

208, 212
Dipyrrlmethane 238, 239

AA

386

UNITY AND DIVERSITY IN BIOCHEMISTRY

Disaccharides 57, 154
Distearopalmitin 61
Disulphide link 276
Diversity, intraspecific 323

specific 317-323
DNA. See Deoxyribonucleic acid
Docosanoic acid 9
Dodecanoic acid 9
Dog 298

Dotriacontanoic acid 9
DPN, DPNH. See Diphosphopyridine-

nucleotide
DPHN -> aldehyde-transhydrogenase,

see Alcohol dehydrogenase
DPNH-cytochrome c-transelectronase

163
DPNH -^ pyruvate-transhydrogenase,

see Lactic dehydrogenase
DPT. See Diphosphothiamine

Earth 3

Eicosanoic acid 9
Elasmobranchii 328, 329
Electrons 1 1 1 , 1 1 2, 1 1 4, 142, 1 62, 1 74,
211

acceptors 211

liberation 207

transfer 142, 145, 162-164, 174
Elements 5

Embden-Meyerhof, see Glycolysis
Endoderm cells 307
Endopeptidases 1 54
Endoplasm 277
Endoplasmic recticulum 276
Energetics, biochemical 131-151

glycolysis 223
Energy 131, 132, 133

activation 151

bonds 137, 138, 139, 142, 144, 159

carbon 351-361

cellular 145

coupling 136

donors 260, 261

electromotive force 134

equilibrium constant 133

food 138

free 131, 133, 135, 227

nuclear 135
Enolase 165, 191, 194
Enolphosphates 137
Entropy 132

negative 149

Enzymes 120,151-171,181,261,277,
278, 280, 301, 303, 308, 309, 312,
323

concentration 1 67

oxido-reduction 162

reaction chains 282-286

substrates 169, 172

systems 341-344

transferring, see Transferases
Equilibrium, chemical 147-149
Ergosterol 41, 236
Eriodictyol 50
Erythrocytes 314
D-Erythrose 15, 22
L-Erythrose 1 5
L-Erythrulose 1 7
Escherichia coli 231, 243, 245, 248, 251,

260, 279, 301
Ester link 276
Esterases 153, 155
Ethanoic acid 9
Ethanol 195
Ethanolamine 221
Ethylallylamine 51
Ethylene-reductase 198, 200
Evolution 324

biochemical 333-345

heteromorphic 336
Exopeptidases 154

FAD. see Flavin adenine dinucleotide
Fatty acids 8-13, 74, 221, 241, 268,
280

biosynthesis 240

branched chain 10

cycle 196, 197, 198, 200, 203, 223,
228, 252

saturated 9

unsaturated 10
Fermentation 186-190, 195, 284
Ferricytochrome 120, 207
FerrihEemoglobin 117
Ferriporphyrim 114, 115
Ferriproteins 1 26
Ferritin 1 26

Ferrocytochrome 120, 207
Ferroporphyrin 112, 113, 294
Fibrocytes 312
Fish 319, 321, 328, 339
Flavan 47, 48
Flavanone 49
Flavin 54

INDEX

387

Flavin adenine dinucleotide (FAD) 71 ,
123, 163, 176, 197, 209, 211, 258,
259

Flavin mononucleotide (FMN), 69,
70, 122, 163, 176, 258

Flavin monophosphate, see Flavin mono-
nucleotide (FMN)

Flavin phosphate (FP) 122

Flavins 59

Flavone 49

Flavonol 49

Flavoproteins 207

Flavoxanthin 35, 37

FMN. see Flavin mononucleotide

Folic acid 55, 242

Food chains 349

Forces, EMF 134
van der Waal 85, 275
vital 273

Formaldehyde 229, 269

Formamide 216

Formamidinoglutaric acid 216

Formyl reactions 229

Formylglutamic acid 216

Formylglycinaminoribotide 255, 256

Formylglycine 242

a-Formylglycyl-D-penicillamine 79

Formylkynurenine 220

FP. See Flavin phosphate

j8-Fructofuranose 57
-1,6-PP, 188
-6-P 188

Fructohexokinase 160

Frxictosans 90

Fructose 17, 22, 58, 67, 89, 159, 161,
191, 232
-1 -phosphate (F-l-P) 66, 159
-1-6-diphosphate (F-1,6-PP) 65,
66, 165, 189, 191, 195, 232, 358

Fructose-6-phosphate (F-6-P) 65, 66,
188, 191, 193, 194, 223, 255, 262

j8-Fructosidase 1 54

L-Fucose 23

Fucosterol 41

Fucoxanthin 37

Fumarase 204

Fumaricacid 12, 199, 201, 207, 219,
224, 231, 245, 310, 311, 366

Fumarylacetoacetic acid 219

Furanose 19

Galactans 90

Galactohexokinese 160
/3-Galactopyranose 58
Galactosamine 22
Galactose 16, 22, 58, 91, 191

-1 -phosphate (Gal-l-P) 160, 166,

191
a-Galactosidase 154
j8-Galactosidase 154, 268
Galactowaldenase 166, 191
Galacturonic acid 92
L-Galaheptulose 18
Gastrophilus 296, 300, 301
Genes 304, 323
Genetic control 286
Genotypes 305, 306, 323
Gentiobiose 154
Globin 155, 293
D-Glucofuranose 20, 21
Glucoketoheptose 18
Gluconic acid 24, 163
D-Glucopyranose 20, 21, 85
a-D-Glucopyranose 20
^-D-Glucopyranose 20
a-Glucopyranose 57
^-Glucopyranose 58
Glucosamine 22, 91
D-Glucosamine 22, 91
Glucosan 21
Glucose-1 -6-diphosphate (G-1 ,6-P P)

66, 166, 175, 191
Glucose oxidase 162
Glucose-6-phosphatase 262
Glucose-1 -phosphate (G-l-P), 65, 67,

160, 166, 175, 188, 191, 195, 225,

254, 262, 263
Glucose-6-phosphate (G-6-P) 65, 66,

123, 146, 159, 163, 175, 187, 193,

195, 223, 253, 255, 262, 308, 360
G-6-P-dehydrogenase 262
Glucose-1 -phosphokinase 160
Glucose, transport 146, 147
a-Glucose 166
a-D-Glucose 18
/3-Glucose 166
jS-Glucose -f Oa-transhydrogenase, see

Glucose-oxidase
/3-D-Glucose 18
L-Glucose 1 5
Glucoses 12, 16, 18, 22, 24, 51, 58, 67,

85, 87, 90, 92, 132, 137, 146, 163,

187, 190, 195, 223, 228, 248, 261,

263

388

UNITY AND DIVERSITY IN BIOCHEMISTRY

a-Glucosidase 154
iS-Glucosidase 59, 85, 90, 154
a-D-Glucosyl-D-glucose 263
^-Glucuronidase 279
Glucuronates 310, 311
Glucuronic acid 24, 66, 163
D-GIucuronic acid 13
Glutamate-alanine transaminase 161
Glutamate-aspartate transaminase 161
D-Glutamic acid 166
L-Glutamic acid 26, 56, 78, 96, 99,
101, 158, 166, 210, 213, 214, 216,
222, 223, 243, 245, 268, 309, 311
Glutamic acid, degradation 215
Glutamic acid dehydrogenase 211,

215
Glutamic-oxaloacetic enzyme 214
Glutamic pyruvic enzyme 214
Glutamic racemase 166
Glutamic y-semialdehyde 244
Glutaminase 157, 214
L-GIutaminase 215
Glutamine 99, 102, 158, 161, 214, 243,

244, 261, 367
Glutathione 78, 164, 261
Glutathione-S-S-glutathione 163
Glycera convoluta 295
D-Glyceraldehyde 15, 16, 163, 254
L-Glyceraldehyde 15, 18, 25
Glyceraldehyde-3-phosphate 252, 254
Glyceric acid 12, 64

phosphoric esters 64
Glycerides 61, 252, 253, 277
Glycerol 23, 61, 74, 163, 189, 191

phosphoric esters 64
a-Glycerophosphate 164
Glycerophosphatides 74
Glycinamideribotide 256
Glycine 27, 78, 79, 96, 224, 229, 240,
242, 268, 367
degradation 221, 224
Glycine-betaine 233
Glycoaldehyde, active 193, 254
-phosphate 193
-transferases, see Transketolases
Glycogen 58, 85, 88, 138, 190, 195,

225, 227, 232, 261-263
Glycol 14, 15
Glycollic acid 12, 229, 269
Glycolvsis 142, 144, 186, 223-230,
269, 284, 285
aerobic and anaerobic 285

reactions 195

terminal stages 194
Glycosidases 1 54
Glyoxylic acid 13,99,211
Guanidoacetic acid 233
Guanine 53, 107
Guanosine 59, 255

monophosphate 256
D-Gulose 16
L-Gulose 16
Gums 91
Guvacine 29
Gramicidines 79
Grana 122, 354
Growth factors 184

pH 114,168,299,317,336
Hasmatins 114
Haemerythrins 125, 126
Haemochromogens 113, 114, 115, 119
Haemocyamins 120, 124, 125, 126
Haemoglobin 46, 115, 117, 120,

292-301, 311, 315, 316, 317-322,

328, 333, 334, 335, 336
Haemopis sanguisuga 295
Hemopoiesis 72
Haemoproteins 111, 119, 120
Haems 112

Haldane effect 117,336
Haptene 302
Harden and Young ester, see Fructose-

1 -6-diphosphate
Heat and energy 131, 132

of combustion 132
Helium 135
Heparin 92

Heptulose diphosphate (HDP), 357
Heptulose monophosphate (HMP) 357
Heredity 286
Hesperidin 49, 50
Heteropolysaccharides 91
Heterosides 57
Heterotrophs 179
Hexacosanoic acid 9
Hexadecanoic acid 9
Hexanoic acid 9
Hexatriacontanoic acid 9
Hexokinases 159, 160, 188, 195, 262
Hexosans 85
Hexosediphosphatase 156
Hexosediphosphates 358

INDEX

389

Hexosemonophosphate shunt, see also
Pentose cycle 186, 191, 223, 340,
359
Hexose phosphates 65, 165
Hill reaction 356, 357
Hippuric acid 260
Histamine 210

deaminase 216
Histidine 27, 46, 96, 104, 210, 224,
229, 243, 250, 251, 265, 269, 366

deaminase 216

degradation 215, 216, 224
Histidinol 250, 251

phosphate 250
Histiocytes 312
Histones 96, 111, 279
Holopolysaccharides 85-91
Holosides 57
a-Holosides 154
|8-Holosides 154
Holothuria 294
Homocysteine 243, 245
Homogentisic acid 219, 224
Homogeny 336

Homoserine 243, 245, 246, 247, 268
Hormones 306, 315, 344

peptide 80
Horse 294, 295, 299, 335, 337
Hyaluronic acid 91
Hyaluronidases 155
Hydrogen 5

acceptor 227, 367

atoms 135

bonds 98, 99, 110, 275

isotopes 183

transfer 162, 163, 174, 176, 183 207
Hydrolases 153, 179
Hydrolysis 167, 173
Hydroperoxidases 120, 164
Hydrosphere 1, 3, 4
/S-Hydroxybutyric acid 12
^-Hydroxybutyryl-CoA 197
^-Hydroxyglutamate-semialdehyde 222
j8-Hydroxy-isovaleryl-CoA 217
Hydroxykynurenine 220
Hydroxylysine 25

/3-Hydroxy-^-methylglutaryl-CoA 2 1 7
Hydroxyphenylpyruvic acids 219
Hvdroxyproline ' 27, 221, 224, 243,
244, 268

degradation 221, 224
Hypoxanthine 53, 240, 256

D-Idose 16
L-Idose 16
Imidazole 47,53,115,250

-glycerol 251

-glycerophosphate 250

-hydroxyacetone 251

-hydroxyacetonephosphate 250

-propionic acid 216

ring 250
a-Iminoglutaric acid 215, 224
a-Iminopropionic acid 212
Indole 43, 46, 220, 248, 250

derivatives 46

-ligase 250

-pyruvic acid 250
a-Indoyl-acetic acid 46
Inheritance 304-316
Inosine 256

monophosphate 256
Inositol 23, 74
Insects 292, 331, 332, 341
Insulin 97, 104, 106, 312

amino acids 106
Intestine, cells 307
Inulin 85, 90
Inulinases 155
Invertase, see /3-Fructosidase
Invertases 58, 154, 169, 254
Invertebrates 290, 292, 321, 338, 339
Ionosphere 1
Iron 111,112,113,117,119,120

protein bound 124
Isoalloxazine, see Flavin
Isobutyryl-CoA 217
Isocitric acid 230, 231
Isocitric dehydrogenase 203, 204
Isoelectric points, haemoglobins 295
Isoleucine 27, 218, 224, 246, 247, 268

degradation 216, 217, 224
Isology 333-336
Isomerases 68, 166
Isomerization 204
Isopentane units 289
Isoprene 30, 235, 236, 237
Isotopes 183
lsovaler\'l-CoA 217

Keratin 330

^-Keratin, X-ray pattern 95
a-Ketoadipic acid 251
a-Ketobutyric acid 247
a-Ketodecarboxylases 165, 212, 232

390

UNITY AND DIVERSITY IN BIOCHEMISTRY

^-Ketodecarboxylases 165, 212
Ketogenesis 309
Ketoglutaraldehyde 222
a-Ketoglutaric acid 13, 214, 218, 202,

203, 204, 208, 211, 215, 218, 222,

224, 230, 231, 243, 267
a-Keto-j8-indolylpyruvic acid 221
a-Ketoisocaproic acid 217, 224, 247
a-Ketoisovaleric acid 217
a-Keto-j8-methylvaleric acid 217, 247
Ketone bodies 241, 308, 309
j8-Ketoreductase 198, 200
Ketoses 15, 17
^-Ketothiolase 198, 200
Kinetics 166

enzyme reactions 283
Knoop jS-oxidation 197
Kuhling's synthesis 54
Kynurenic acid 220
Kynurenine 220

Lactic acid 11, 12, 141, 163, 173, 195,

223, 225, 262

oxidation 141
Lactic dehydrogenase 162, 173, 174
L-Lactic acid 195
Lactose 58, 154
Lama s.p. 321
Laminaria 89
Laminarin 89
Lanoline 62
Lecithins 74, 252, 253, 276

a and j8 74
Legumes 361-363
Leucocytes 314
D-Leucine 79

L-Leucine 27, 213, 217, 222, 246, 247,
268, 294

degradation 216, 217, 224
Leuconostoc dextranicutn 89

mesenteroides 89
Levans, see Fructosans
Levoglucosan 21
Lichenin 89
Licheninase 155
Life 5

Light, see Photoreception
Limulus 1 20

Linkage, see also Bonds 110, 139-144,
154, 275

disulphide 276

glycoside 1 54

polypeptide 77-80, 260, 276

salt 84, 276
^-Linkages 263
Linoleic acid 10
Linolenic acid 10
Lipids 338

complex 61, 74-76, 127, 252, 278

ternary 61
a-Lipoic acid, see Thioctic acid
Lipoproteins 127
Lipothiamide pyrophosphate (LTPP)

196, 205
Lithosphere 1, 3, 372
Liver cells, 308, 309, 310
Lobster, spiny 120
Lohmann reaction 159
LTPP. See Lipothiamide pyrophosphate
Lumbricus 294
Lumichrome 60
Lumiflavin 55, 60
Lutein 37
Luteose 92
Lycopene 31, 37

trans and cis 304
Lycopyll 33

Lycopersicon escidentiim 304
Lycoxanthin 33

Lysine 27, 96, 102, 210, 219, 224, 251,
252, 265, 268, 294

degradation 218, 224
Lysosomes 279

Macromolecules 61, 83-127, 261-269,
339

proteins 301—303
Magnesium 5

complexes 1 22
Malate-fumarate transformation 201
Malate-oxaloacetate reactions 225
Maleic acid 11
Malelylacetoacetic acid 219
Malic acid 12, 51, 141, 203, 213, 223,

228, 231
Malic dehydrogenase 201, 204
Malic enzyme 213
Malonicacid 11,52,199
Maltase, see a-Glucosidase
Maltose 58, 154

phosphorylase 158
Man 296, 298, 299, 318
Mannans 90
D-Mannoheptulose 18

INDEX

391

Mannose 16, 92
D-Mannuronic acid 85
Mass action, law 133
Membranes 146
Mesobilierythrin 291
Mesobiliviolin 291
Metabolism 144, 182, 309

biosphere 349-365, 377-379

cellular 282
Metalloflavoproteins 122
Metalloproteins 111, 123
Metaphosphatases 156
Methacryl-CoA 217
Methaemoglobin 117, 294
Methanoic acid 9

Methionine 232, 233, 243, 245, 246,
247, 265, 268

active 161
Methyl groups 154, 161, 230, 232-235

cycle 234
3-Methylbutanoic acid 10
L-Methylbut^'r>-l-CoA 217
j3-Methylcrotonic acid 236
5-Methylcytosine 107
Methylglyoxal 188
Methylnicotinamide 161, 232
Micrococcus aureus 283, 367
Micelles 84, 86
Michaelis constant 169-171
Micro organisms 184, 185

carbon cycle 371—374

nitrogen cycle 375

sulphur cycle 376
Microsomes 266, 277
Milieu 315-316
Mitochondria, see also Lysosomes 274,

278, 285
Mitosis 280

Molecular weight, chemical and physi-
cal 83, 84

globular protein 97

haemoglobins 295
Mononucleotides 67
Moulds 251
Mucilages 91
Mucoitinsulphates, see Polysaccharide

sulphuric esters
Mucopolysaccharidases 155
Mucopolysaccharides 22, 91
Muscle cells 312
Mutants 184, 185
Mutarotase 1 66

Mutarotation 18, 20

Mutatochrome 32

Mycobacterium tuberculosis 92

Myoglobin 295

Myokinase 160

Myxine 295

Mjrxophycas 364

Myxoxanthin 34

Nereis marina 295

Nervous system 307

Neuberg ester, see Fructose-6-phos-

phate
Neurones 307

Neurospora 218, 219, 245, 246
Nicotinamide 51, 233, 258, 259

mononucleotide 70, 161, 260

riboside 259
Nicotine 51

methyl transferase 161
Nicotinic acid 51, 220, 221, 260
Nitrates 362
Nitrobacter 352, 375
Nitrogen 2, 3, 5

cycle 372-376, 379

equilibrium 181

fixation 362-363

in proteins 100
Nitrosococais 352
Nitrosomonas 352, 375
Nostoc 364
Normoblasts 314
Notomastus 295

Nucleic acids 107, 265, 266, 267
Nucleons 183
Nucleoproteins 1 07-1 1 1
Nucleoside-phosphorylase 255
Nucleoside-N-transglucosidase 255
Nucleosides 59, 161, 255
Nucleotides 67-72, 110, 255
Nucleus 274, 279-280

Oceans 3, 4, 377, 379
Octacosanoic acid 9
Octadecanoic acid 9
Octanoic acid 9
Octatriacontanoic acid 9
Oenin chloride 50
Oestrane 43
Oestrone 43

Old yellow enzyme 122, 123
Ontogenesis 305-315
Optical activity 166

392

UNITY AND DIVERSITY IN BIOCHEMISTRY

Optical isomers 30
Orbitals 112
Organelles 274
Organisms 5, 306
Organs 181

Ornithine 25, 158, 244, 245, 309, 311,
368

cycle 309-311
L-Ornithine 210
Orotic acid 257
Orotidine 241, 257
Orthophosphoric acid, diesters 74

esters 62
Osides 57-59, 152, 161, 254
Osteoblasts 313
Oxaloacetate-malate system 201
Oxaloacetic acid 13, 203, 204, 214,

224, 230, 231, 267, 363, 364
Oxalosuccinic acid 13, 203
Oxalosuccinic decarboxylase 204
jS-Oxidation 196
Oxidoreductases 162-164
Oxido-reduction, potentials 138, 140,
141

reactions 142, 144, 145
Oximino succinic acid 363
Oxychlorocruorin 118
Oxygen 2, 3, 5

transport 320-322
Oxygenation, haemoglobins 296-301,

317-322, 336
Oxyhaemerythrin 1 26
Oxyhsemoglobin 116, 117, 295, 336
Oxytocin 80, 81
Ozonosphere 1, 3

Palmitic acid 228
Palmityl-CoA 253
Pancreas, cells 308
Pantetheine 259, 260
Pantothenic acid 79, 259, 260

4'-phosphate 72
Parahasmatins 114, 115, 117
Paramagnetism 113
Parathyroid gland, cells 311, 312
Pasteur effect 285
Pectic acid 90
Pectins 90, 369
Penicillin G 80
Penicillins 80
Penicilliiirn 80, 244

Penicillum charlesii 92

luteum 92
Pentanoic acid 9
Pentosans 90
Pentose 358

cycle, see also Hexosemonophosphate

shunt 191, 193, 255, 356-359
phosphates 226, 253
Pentoses 241, 268
Peptidases 153, 154, 279
Peptides, bonds, see Bonds, peptide
natural 77-82
hormones 80
hydrolysis 173
insulin 106
synthetic 77-78
Perhydrophenanthrene 38
Peroxidases 46, 120, 121
PGA. see 3-Phosphoglyceric acid
PGAD. see Phosphoglyceraldehyde-

dehydrogenase
Phasophorbide 122
Phaeophytin 122
Phalloidin 80
Phascolosoma elongatum 120
Phenanthrene, natural derivatives 38
Phenolase 123, 124
complex 302, 303
Phenoloxidases 164
Phenolpyrrol 43
Phenotype 315-316
Phenylalanine 28, 210, 213, 219, 222,
229, 248, 249, 269
degradation 219, 224
L-Phenylalanine 210
Phenylglucosazone 22
Phenylisothiocyanate 104
Phenylpyruvic acid 249
Phenylthiocarbamylpeptide 104
Phosphatases 153, 155-156, 357
Phosphates 187, 377
acceptors 285
cycle 138

group transfer 188-190
Phosphatidic acid 252, 253
Phosphatides 276
Phosphatidylcholines, see Lecithins
Phosphatidylethanolamines, see Cepha-

lins.
L-Phosphatidylserine 74, 75
Phosphoamides 63
Phosphoarginine 63, 159

INDEX

393

Phosphocreatine 63, 159, 160
Phosphodiesterases 156
Phosphodihydroxyacetone, 64, 163,

165, 189, 191, 195
Phosphoenolpyruvic acid 143, 190,

195
Phosphofructokinase 161, 188, 195
Phosphoglucoisomerase 188, 195
Phosphoglucomutase 166, 195, 262
Phosphogluconic acid 163, 192, 226,

360
Phosphoglyceraldehyde 65, 142, 161,

165, 189, 191, 193, 195, 267
oxidation 142

Phosphoglyceraldehyde dehydrogenase

(PGAD) 142, 190, 195
2-Phosphoglvceric acid 64, 143, 166,

175, 190, 195
3-Phosphoglyceric acid (PGA) 64, 143

166, 175, 190, 195, 226, 232, 284,
357, 360

3-Phosphoglvceric phosphokinase 144,
190, 195

Phosphoglyceromutase 166, 195
system 175

Phosphohexokinase 160, 195

Phosphoinositides 75

Phosphokinases 159

Phospholipides 309

Phosphomonoesterases 155

Phosphomutase 255

Phosphopentokinase 208

Phosphoribomutase 166

5'-Phosphoribosylamine 256

Phosphoribosylpyrophosphate (PRPP)
255

Phosphoric acid, natural esters 62

Phosphoric ester bridge 108

Phosphoroclastic reaction 229

Phosphorus 5, 361, 377

Phosphorus circulation 377, 379

a-Phosphorylase 158

Phosphorylases 158, 161, 191, 195,
262

Phosphorylated compounds 137

Phosphor>'Iation 142, 144, 207, 208,
225, 228, 262, 285

Phosphorylcholine 252

Phosphorylcytidyl transferase 253

5-Phosphoshikim.ic acid 249

Phosphosphingosides, see Sphingomye-
lins

Phosphotransferase 266
Phosphotriose 189
Photoreception 290
Photosynthesis 145, 146, 291, 340,

351, 354-361, 371, 377
Phycobilins 291, 325
Phycocyanins 291
Phycoerythrins 291
Phytofluene 304
Phytol 31, 121, 122
Phytonionas tiunefaciens 89
Pig 298
Pigeon 295, 319
Pigments 48, 50, 119

quinoid 38
Pipecolic acid 29, 218, 224
Pitocin 344
Pituitary 306
Planorbis 295, 300, 301
Plants 290, 292, 302, 354, 361
Plasmalemma 277
Plasmalogens 75

Polygalacturonic acid, see Pectic acid
Polyglucosamines 91
Polyglucoses 89
Polymers, see Macromolecules
Polypeptides, amino acid sequence 104,
' 105

chains 274, 275, 276

a-helical chains 97

hydrogen bridges 99

ribbon 94, 95
Polyphenols 124
Polysaccharases 1 54
Polysaccharide sulphuric esters 92, 93
Polysaccharides 85, 261-263

bacterial 92

blood group 92

pneumonococcal 92
Polyuronides 90
Porphin 43
Porphobilogen 238
Porphyrins 44-46,112,222,238,239,

267, 291-301
Porphyrospin 291, 337, 343
Potassium 5
Precursors 184
Pregnane 42
Prephenic acid 248, 249
Progesterone 42

Proline 28, 46, 96, 97, 104, 222, 243,
265, 268

394

UNITY AND DIVERSITY IN BIOCHEMISTRY

degradation 221, 224
Propionyl-CoA 217, 224
Protamine 96, 111

Proteins 83, 93-104, 123, 124, 172,
263-269, 336

amino acid incorporation 267

composition 100, 103, 105

denaturation 98, 100

fibrous 94

globular 96, 97

hydrolysate, analysis 103

macromolecules 301-303

metabolism 309

reactive groups 101

residual 111

structure analysis 93-102

synthesis 263-269

titration 100
Protium 183
Protohsm 113, 293
Protons, see also Transhydrogenases

174, 183, 207
Protoplasm 274
Protoporphyrin 45, 46, 117, 291
Protopteriis 337
PRPP. see Phosphoribosylpyrophos-

phate
L-Psicose 16

Pteridine derivatives 55-56
Pterins 55
Pteroic acid 56

Pteroyl glutamic acid, see Folic acid
Purine 53, 107, 221, 267

derivatives 53

nucleotides 255, 258

ring 240, 255
Putrefaction 46
Putrescine 210
Pyran 47, 48
Pyranose 19
Pyrazine 52
Pyridazine 52
Pyridine 51, 71
Pyridoxal 52, 242

-5 -phosphate 66, 174, 220
Pyridoxamine 52

-5-phosphate 66
Pyridoxine 52
Pyrimidine 52, 55, 107, 221, 241, 268

derivatives 52-54

nucleotides 255, 258

ring 241, 255

Pyrophosphatases 137, 156

Pyrophosphates 137, 161, 235
bonds 179

Pyrrole derivatives 43

Pyrrolidine 46, 72

id'-Pyrroline-5-carboxylic acid 244

Pyruvic acid 13, 137, 141, 143, 159,
163, 190, 194, 196, 212, 221, 222,
224, 226, 228, 229, 230, 231, 247,
262, 267, 269, 280, 355
oxidative decarboxylation 196, 203

Pyruvic carboxylase 165

Pyruvic decarboxylase 196

Pyruvic phosphokinase 144

Q-enzyme 263
Quercetin 49
Quercitrine 50
Quinic acid 249
Quinolinic acid 220

Racemases 166
Rackers reaction 357, 358
Raia ocellata 296, 299, 318, 321
Reaction chains, enzymic 282-285
Reactions, priming 186-228

biosphere 179-228

endothermic and exothermic 131,
132

exergonic 136

metabolic 144, 182, 309, 349-365,
377-379

oxidation-reduction 142, 144, 145

reversible 147-149
Redox potential 140
Regulation, cellular 282-286
Reptiles 318, 319, 329, 330, 337, 339,

340, 341
Resins 289
Respiration 144, 199
Respiratory chains 207, 208, 209
Respiratory cycles 317-322
Retinene 290, 343
Rhamnose 23
Rhizobium 363, 364
Rhizopus tritici 368
Rhodopsin 290, 337, 343
Rhodoviolascin 33
Rhodoxanthin 36, 37
Ribitol 23, 58

phosphate 66
Riboflavin 59, 259

INDEX

395

j8-D-Ribofuranosides 108

Ribonuclease 103, 156

Ribonucleic acid (RNA) 107, 108,

265, 266, 267, 269, 277, 279, 313
Ribose 60, 70, 221

-1 -phosphate 65, 166, 240, 260
-5-phosphate 161, 166, 193, 253,

254, 359
D-Ribose 16, 22, 58, 70, 107, 221
L-Ribose 16
D-Ribulose 17, 22, 358
L-Ribulose 17
Ribulose-5-phosphate (Ru-P) 161,

166, 192, 193, 194, 254, 357
Ribulosediphosphate (RuPP) 357, 358,

359, 360
RNA. see Ribonucleic acid
Robinson ester, see Glucose-6-phosphate
Rubichrome 34
Rubixanthin 33, 37

Sabellariides 334

Saccharase 154

Saccharic acid 24

Saccharomyces cerevisice 186

Salt linkages, see Linkages, salt

Saponins 289

Sarcosine 232

Sea water 2

Sedoheptulose 193, 226, 254, 267, 268

-1,7-diphosphate 248

-7-phosphate 161, 193, 194, 226,
254
D-Sedoheptulose 18
Senecioyl-CoA 217, 222, 224, 226
Serine 25, 28, 75, 212, 220, 221, 224,
229, 241, 243, 268, 269, 367

degradation 221
L-Serine 25
SH-enzymes 243
SH-glutathione 163
Shemin cycle 221
Shikimic acid 248, 249
Siderophilin, see Transferrin
Slater factor 208
Snakes, see also Reptiles 341
Sodium 5

Soil, ammonification 366-368
D-Sorbitol 23
D-Sorbose 17
L-Sorbose 17
Specialization 341

Species 317, 334
Spermaceti 61
Sphingolipides 75
Sphingomyelins 75, 76
Sphingosine 75, 76
Spionids 334

Spirographis spallanzanii 1 20
Squalene 237, 238
Staphylococcus aureus 264
Starch 87, 369
Stearic acid 83, 198
Sterane 38
Stereoisomerism 29
Sterides 62
Steroids 267

biliary 330

hormones 315

17-keto 42

natural 42

ring 236
Sterols 40-42, 337, 338

in algas 326
Stratosphere 1
Streptococcus fcecilis 367

hcemolyticus 367
Substrates, enzyme complex 169, 172
Succinate-glycine cycle 223
Succinic acid 11, 140, 160, 161, 199,
200, 201, 202, 203, 208, 221, 226,
228, 231, 243, 311
Succinic dehydrogenase 163, 199, 201,

204, 208, 279
Succinyl-CoA 160, 161, 206, 221, 223,
224, 225, 251, 252, 267, 268

-transphosphorylase 160
Sucrose 58, 67, 154, 161, 254

phosphorjdase 158
Sugars 7, 14-24, 57

amino 22

biosynthesis 241

deoxy 22

natural 22

oxidation 24

phosphorylated 65

synthesis 351-353
Sulphur 5, 360, 364

bacteria 352, 353

cycle 376

in proteins 101
Sulphuretum 376
Sun, energy 135, 145

396

UNITY AND DIVERSITY IN BIOCHEMISTRY

Tagatose 1 7
D-Talose 16
L-Talose 1 6
Taraxanthin 37
Tartronic acid 12
Taurine 223
Taxonomy 317-332
Terpenes 30, 31, 267, 289-291
Tetracosanoic acid 9
Tetradecanoic acid 9
Tetratriacontanoic acid 9
Tetrosephosphates 357-360
Tetroses 1 7
Thalassema neptuni 301
Thermobacteriuni acidophilus 265
Thermodynamics, equilibrium 152

laws 131, 132
Thiamine 53, 66

dehydrogenase 164

pyrophosphate 47, 67
Thiazole 47, 53, 66

derivatives 47
Thiobacillus denitrificans 352, 372

thiooxydans 352

thioparus 352
Thioctic acid 47, 146, 208, 359
Thioesters 137
Thioethanolamine 72
Thiophosphates 137
Thiorhodaceae 360
Threonine 28, 30, 222, 229, 245, 247

265, 269
D-Threose 15
L-Threose 1 5
Thymine 52, 107
Thyone 295
Thy one briar ens 295
Thyroid cells 312
Thyronine 52,107,111,255
Thyroxine 228
Tigyl-CoA 217
Tissue, slices 181

connective 312
Titrations, protein 101, 102
a-Tocopherol 48
/8-Tocopherol 48
Tomato 304
Topobiochemistry 284
Torularhodin 36

TPN, TPNH. See Triphosphopyri-
dinenucleotide

TPNH->02-transhydrogenase, see

yellow enzyme
Trace elements 5
Transacylases 154, 161
Transadenylases 161-163
Transaldolase 193, 194
Transaldolization 254
Transaminases 160, 214
Transaminations 213, 311
Transapartases 1 62
Transdeaminations 214
Transdehydrogenases 72
Transelectronases 163, 164
Transferases 159, 162
Transferase systems 154
Transferrin 1 26

Transglucosidase 154, 158, 254, 263
Transglucosidation 255
Transglutamases 1 62
Transhydrogenases, see also Protons

162, 163
Transketolases 161,193,254,357
Transketolization 254
Transmethylases 154, 161
Transmethylations 232-235
Transpeptidases 154, 161
Transphosphatases 159
Transphosphorylases 159, 160, 188
Transport, glucose 146
Transsulphurases 161
Transuridylases 161
Trehalose 154
Triacosanoic acid 9
Tricarboxylic acid cycle 199-207, 215

216, 224, 225, 228, 268, 280, 310,

360
Triglycerides 253

Trimethylglycine, see Glycine-betaine
Triosephosphate dehydrogenase 103
isomerase 1 66
lyases 166
Triosephosphates 64, 189, 226, 231,

232, 357
Triphosphopyridinenucleotide (TPN,

TPN + ,TPNH) 70,146,163,176,

191, 208, 213, 225, 231, 258, 278,

280, 301, 355, 360
Trisaccharides 1 54
Tritium 1 83
Troposphere 1, 2
Tryptophan 28, 43, 46, 79, 96, 111,

132, 138, 229, 248, 250, 267

INDEX

397

degradation 220, 221
desmolase, see Indole-ligase

Tuberculin 92

Tubifex 300

Tyramine 210

Tyrocidines 80

Tyrosine 28, 96, 101, 209, 219, 223,
229, 248, 249
degradation 219, 224

L-Tyrosine 210

UDP. See Uridine diphosphate
UDPG. See Uridine diphosphate glu-
cose
UMP. See Uridine monophosphate
Uracil 52, 68, 107, 255
Urea 52, 77, 158, 166, 182, 329, 368
Urease 152, 158, 167, 367
Urechis 300, 319, 322, 336
Ureidosuccinic acid, see Carbamylas-

partic acid
Uric acid 342
Uricolysis 341, 342
Uricolytic enzyme system 342
Uridine 68, 241, 255

diphosphate (UDP) 68, 161, 311

diphosphate glucose (UDPG) 68,
69, 161, 191

diphosphateglucuronate (UDP-glu-
curonate) 310

monophosphate (UMP) 68, 257

phosphates 67

triphosphate (UTP) 64, 68, 161
Urocanic acid 216, 224, 366
UTP. See Uridinetriphosphate

Valency, degradation 216
heteropolar 275
homopolar 276

Valine 28, 79, 217, 222, 246, 247, 269

degradation 216, 217, 224
Van der Waal forces 275
Vasopressin 80, 81, 82
Vertebrates 290, 292, 294, 296, 297,

298,321,327,337,338,339,344
Violaxanthin 35, 37
Vision 290
Vitamin A, Aj, A^ 290
Vitamin B. 60
Vitamin Bj,, see also Cyanocobalamin

46, 63

Water 5

Waxes 6 1

Work, and energy 131, 132, 133

cellular 146
Worms 293

X-ray diffraction, fibrous protein 95
Xanthine 53

oxidase 72, 162

— 'Oj-transhydrogenase 162, 163
Xanthophyll 34, 37, 121
Xanthophyllepoxide 35
Xanthophylls 290, 325
Xanthosine monophosphate 256
Xanthurenic acid 220
Xylans 90
Xylulose 17, 193

Yeast 186, 188
Yellow enzyme 69, 164

Zeaxanthin 34, 37
Zona fasciculata 315