The nitrogen metabolism of micro-organisms

THE NITROGEN

METABOLISM OF

MICRO-ORGANISMS

B. A. FRY

! METHUEN'S MONOGRAPHS ON
i^&.iJIOCHEMICAL SUBJECTS

]\ Marine Biological Laboratory Library

I Woods Hole, Mass.

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Presented by

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METHUEN'S

MONOGRAPHS ON

BIOCHEMICAL SUBJECTS

General Editors : SIR RUDOLPH PETERS, f.r.s.
and F. G. YOUNG, f.r.s.

THE NITROGEN METABOLISM
OF MICRO-ORGANISMS

The Nitrogen Metabolism
of Micro-organisms

B. A. FRY,

B.A., Ph.D.

Lecturer in Microbiology

in the University of Sheffield

WITH 3 PLATES & 14 DIAGRAMS

LONDON: METHUEN & CO. LTD
NEW YORK: JOHN WILEY & SONS, INC.

First published in ig55

I.I

CATALOGUE NO. 4141/u (mETHUEN)

PRINTED AND BOUND IN GREAT BRITAIN
BY BUTLER AND TANNER LTD., FROME AND LONDON

PREFACE

Little reflection is required to realize that nitrogen is a
constituent of numerous compounds of biological interest,
and all acquainted with present-day biochemistry are aware
that during the last ten years the former emphasis on the
study of the degradation of complex substances has been
largely replaced by an active interest in mechanisms of syn-
thesis, and in particular, in the synthesis of proteins and the
metabolic role of the nucleic acids. Micro-organisms are
proving to be of great value in the unravelling of the routes
whereby amino-acids, nucleotides and other compounds are
synthesized in mvo, and for a long time they have been used
with great success in experiments designed to elucidate the
functions of the many water-soluble substances now in-
cluded in the B group of vitamins.

In this monograph an attempt has been made to survey
as comprehensively as possible the nitrogen metabolism of
micro-organisms and to treat the subject in such a manner
as to reflect current trends in modern microbiology. The
monograph is based on a series of lectures given in a one-
year post-graduate course of microbiology held in the Uni-
versity of Sheffield, and it is hoped that advanced students
at other universities and research workers in allied fields will
find it a convenient and concise introduction to one impor-
tant section of microbial biochemistry. If it be thought that
some topics receive more attention than they warrant, then
the author accepts full responsibility for his choice and de-
fends it on the grounds that these topics either encompass
ideas of wider significance or serve to focus attention on how
little has really been established. Though the title of the
monograph is all-embracing and in the text examples are
drawn from experiments with bacteria, fungi, algae and pro-
tozoa, the m.ain emphasis is naturally on the first two of these
four groups, since most work has been done with species of
bacteria and yeasts. There is not space to mention every

vi NITROGEN METABOLISM

organism which has been studied, but the reader's search
for additional information should be aided by the books re-
commended for general reading and the detailed biblio-
graphy appended to each chapter.

It is with very great pleasure that I record my thanks to
Dr. S. R. Elsden for his interest in the preparation of this
monograph and to him and Dr. J. L. Peel for reading the
drafts of the various chapters and making many helpful sug-
gestions. I am also grateful to Dr. E. F. Gale, F.R.S., for
reading the completed manuscript.

Sheffield

1953

ACKNOWLEDGMENTS

I AM indebted to the following authors, editors and pub-
lishers for their permission to reproduce figures which have
appeared in the literature: Fig. 3.1, Prof. A. L. Audus and
Fig. 10.2, Dr. P. Mitchell, and the Editors of Nature; Fig.
3.2, Prof. J. H. Quastel and Dr. P. G. Scholefield, and
Messrs. Williams and Wilkins Co., U.S.A.; Fig. 4.1, Prof.
P. W\ Wilson and the Editors of the Biochemical Journal',
Figs. 6.1 and 6.2, Dr. E. F. Gale, F.R.S., the Academic
Press Inc., U.S.A., and the Editors oi t\\t J ounml of General
Microbiology; Fig. 8.1, Prof. L. Gorini and Prof. CI. Fro-
mageot, and the Elsevier Publishing Co., Inc.; Plate I, Dr.
B. Davis and the Editors of Experientia; Plate III, Prof. R.
Tulasne and Dr. R. Vendrely, and the Long Island Bio-
logical Association: Plate II is a photograph of a chromato-
gram kindly prepared by Dr. R. Markham.

CONTENTS

CHAP. PAGE

PREFACE V

I INTRODUCTION I

II AMINO- ACID CATABOLISM lO

III NITRIFICATION AND DENITRIFICATION 32

IV THE FIXATION OF NITROGEN 45

V SYNTHESIS OF AMINO-ACIDS 6o

VI ABSORPTION OF AMINO-ACIDS BY MICRO-ORGANISMS 8o

VII PEPTIDES AND PROTEINS 95

VIII PROTEOLYTIC ENZYMES 112

IX NUCLEOTIDES AND NUCLEIC ACIDS 1 26

X MODE OF ACTION OF CHEMOTHERAPEUTIC AGENTS I45
INDEX 159

PLATES

FACING PAGE

I SYNTROPHISM AMONG ARGININE REQUIRING

MUTANTS OF ESCH. COLI 69

II ULTRAVIOLET PHOTOGRAPH OF CHROMATOGRAM

SHOWING SEPARATION OF PURINES AND PYRIMI-
DINES IN YEAST NUCLEIC ACID I31

III PHOTOMICROGRAPHS OF A COLON BACILLUS BEFORE

AND AFTER TREATMENT WITH NUCLEASES 1 33

ao9o6

CHAPTER I

INTRODUCTION

Energetics of biological systems

In recent years the attempts to analyse the energetics of
biological systems in terms of established thermodynamic
principles have naturally focused much attention on the
reactions in such systems which yield energy and those
which utilize energy [6]. When energy is supplied to or
liberated in a system, there are limitations regarding the
conversion of one form of energy into another (Second Law
of Thermodynamics). In other words, only part of the
energy content of any system is available for doing further
work, and this useful energy is termed /r^^ energy. Chemical
reactions in which there is an output of free energy are
described as exergonic and those in which there is an uptake
of free energy as endergonic. Reproduction, growth and the
maintenance of life are all endergonic processes and are
therefore intimately associated with mechanisms able to
supply them with energy.

It is generally believed that energy becomes available in
biological systems as the direct or ultimate result of oxida-
tion reactions [8, lo]. The oxidation of one substance must
necessarily be accompanied by the reduction of another
and a biological oxido-reduction reaction involves the trans-
fer of hydrogen atoms or electrons [14]. Consequently the
substance which is oxidized is sometimes described as being
a hydrogen donor [H-donor], whilst the one being reduced
is termed the hydrogen acceptor [H-acceptor]. The com-
plete oxidation of any one substance proceeds by one or
more simple steps, each catalysed by the appropriate
enzyme, and in all known reactions the transfer of hydrogen
atoms or electrons to the ultimate H-acceptor is effected by
one or more intermediate carriers. In aerobic organisms,
molecular oxygen serv^es as the H-acceptor and, according
to the enzyme concerned, the end-product is water or

2 NITROGEN METABOLISM

hydrogen peroxide. Organisms which by chance or by
necessity are Hving in an anaerobic environment must use
a substance other than oxygen for this purpose. Such a sub-
stance may be derived from the environment (e.g. CO 2 ,
nitrate or acetate) or may be a product of the organism's
cataboHsm (e.g. in the lactic acid bacteria, pyruvate is
reduced to lactate).

The esterification of inorganic orthophosphate is an
integral part of the mechanism whereby endergonic reac-
tions are able to utilize the energy made available by oxido-
reduction reactions. Our conception of this mechanism is
mainly due to Lipmann [10], who pointed out that phos-
phorylated compounds can be divided into two groups
according to the amount of energy released by their hydro-
lysis: some yield about 3,000 cal. per mole whilst others
liberate 10,000 to 16,000 cal. per mole. Lipmann proposed
that the latter should be known as high-energy (or energy-
rich) phosphate compounds and that they contain what he
termed high-energy (or energy-rich) phosphate bonds, the
hydrolysis of which yields 10,000 or more calories of free
energy per mole of inorganic orthophosphate liberated. The
significance of certain biological oxido-reduction reactions
lies in the fact that they are associated with the formation of
energy-rich phosphate bonds: these arise either during the
actual oxidation of the organic substrate or else during
the transfer of hydrogen (or electrons) to a H-acceptor. In
the former case oxidation of the organic substrate is accom-
panied by its esterification with inorganic orthophosphate
and in consequence most of the energy made available by
the oxidation reaction is not liberated as heat but is retained
in the oxidized substrate in association with the newly in-
corporated phosphate group. The only known example of an
energy-rich phosphate group arising by a non-oxidative re-
action is found in e«o/-2-phosphopyruvic acid, a substance
formed by the dehydration of 2-phosphoglyceric acid under
the influence of enolase. The phosphate groups and their
associated energy can be transferred, in the presence of the
appropriate enzyme (a phosphokinase), to adenosine diphos-
phate (ADP), or sometimes to adenosine monophosphate

INTRODUCTION 3

(AMP), thus forming adenosine triphosphate (ATP) or
ADP respectively.

In a biological system, the only known way in which the
energy released by an exergonic reaction can be made
available to an endergonic reaction, is for the two reactions
to be coupled together by means of a substance which
participates in both. This is the function of ATP, which by
virtue of its high-energy phosphate groups acts as an energy
carrier between reactions yielding energy and those utilizing
energy. Adenosine triphosphate participates in the latter by
reacting with, and thus activating, one of the reactants, and
by this means the total free-energy content of the reactants
is raised to a value at least approximately equal to, and often
far greater than, that of the products. From the standpoint
of energy relationships, the conditions are now such as to
favour formation of the products, and the utilization of
ATP in this manner is accompanied by the appearance of
inorganic orthophosphate.

Although it is generally accepted that the energy meta-
bolism of all organisms is associated with energy-rich
phosphate bonds, little is known about how they are formed
except during the anaerobic catabolism of glucose and
pyruvate. The results of contemporary research indicate
that co-factors containing thiol groups probably play an
important role both in the production of energy-rich phos-
phate groups and in their utilization, and that the synthesis
of thiol esters may be an essential intermediate stage in these
reactions (cf. the role of glutathione in the triosephosphate
dehydrogenase system [16], and coenzyme A (Co. A) in the
synthesis of citric acid and other compounds [2]). A sub-
stance having the properties of ATP is believed to be present
in all organisms and ATP has in fact been isolated from
yeast [cf. 4], green plants [i] and animals, but its occurrence
in bacteria is based more on inference rather than its isola-
tion in a pure state [3, 7, 9, 11, 12, 13, 15].

Nutrition: general aspects

Irrespective of the organism, the continuance of life and
the synthesis of cytoplasm are dependent on the availability

4 NITROGEN METABOLISM

of the same basic materials, namely, mineral salts, water and
sources of carbon and nitrogen together with a mechanism
providing energy in a form that can be utilized in biological
systems. Autotrophs are organisms whose carbon require-
ments are entirely satisfied by CO 2 (perhaps in some by
CO). On the other hand, heterotrophs require a more com-
plex carbon source, i.e. an organic compound, as well as
CO 2 . Moreover, heterotrophs usually derive their energy
by catabolism of the organic carbon source and are therefore
to be contrasted with autotrophs which obtain their energy
either from light (photosynthetic autotrophs) or by the
oxidation of inorganic substances, e.g. H2S, S, NagSaOa ,
NHt, NOi", H2 or Fe"*""^ (chemosynthetic autotrophs).
Each chemosynthetic autotroph oxidizes one specific com-
pound, or in certain cases, a limited number of chemically
related compounds, and presumably part of the energy
released during these oxidations becomes available in the
form of energy-rich phosphate groups. How the light energy
absorbed by the chlorophyll of photosynthetic organisms
becomes converted into a form that can be utilized in
enzymic reactions is not yet known, though recent experi-
ments have provided some indications of a possible
mechanism [17].

All autotrophs derive their nitrogen from an inorganic
source and, depending on the organism, use molecular Ng ,
NHt J nitrate or nitrite. Although one or more of the latter
may serve as a complete source of nitrogen for certain
heterotrophs, the nutritional requirements of many of these
organisms are not so simple. It appears that such hetero-
trophs are unable to synthesize one or more of the organic
constituents of cytoplasm and they are therefore only able
to grow if these substances are present in their environment,
i.e. they are exacting towards these substances. The ability
to synthesize complex organic nitrogenous compounds is
especially variable, and whilst some organisms are exacting
towards only one compound, e.g. Salmonella typhosa to
tryptophan and Proteus vulgaris to nicotinic acid, the nutri-
tion of other heterotrophs is far more complex, e.g. Leuco-
nostoc mesenteroides P-60 requires eighteen amino-acids and

INTRODUCTION 5

at least eleven growth factors. (The term growth factor is
used here in the same sense as vitamin in animal nutrition.)
With regard to the amount of carbon used for the synthesis
of cellular material, the contribution of the organic com-
pound serving as a source of carbon and energy varies in-
versely with the number of cytoplasmic constituents which
the heterotroph derives preformed from the environment:
in a rich medium this compound may function primarily as
a source of energy. At one time, autotrophs were diiferen-
tiated from heterotrophs on two counts: firstly that hetero-
trophs were unable to incorporate the carbon of CO 2 into
organic molecules, and secondly that autotrophs live entirely
and exclusively at the expense of inorganic substances.
There is now adequate information to show that both of
these statements require modification [18, 19, 5]. The
growth of heterotrophs is in fact dependent on the presence
of CO 2 and they are known to possess enzyme systems
accomplishing its fixation: but, although essential, CO2 is
neither a complete nor a major source of carbon for hetero-
trophs. Furthermore, it has been established that several
organisms regarded as autotrophs can exist heterotrophic-
ally. For example, in the presence of a suitable H-donor, the
purple sulphur bacteria (Thiorhodaceae) obtain their energy
from light, whilst CO2 and NH3 (or N2) serve as complete
sources of C and N. The H-donor may be an inorganic form
of sulphur or an organic substance such as a fatty acid, and
the Thiorhodaceae can therefore be regarded as facultative
autotrophs. On the other hand, the green sulphur bacteria
use only an inorganic H-donor and appear to be obligate
autotrophs. The Athiorhodaceae (non-sulphur purple bac-
teria) require certain growth factors and usually an organic
H-donor, i.e. they are heterotrophs, although they too-
derive their energy from light.

Synopsis of monograph

Many organisms can derive their energy either directly
or indirectly from nitrogenous compounds, and examples
of this feature of their metabolism are given in separate:

6 NITROGEN METABOLISM

chapters devoted firstly to the fermentation and oxidation
of amino-acids by heterotrophs, and secondly to the auto-
trophic nitrifying bacteria. It will be seen that in nature the
ammonia produced during the decomposition of amino-
acids may suffer one of three fates: (i) oxidation by the
nitrifying bacteria to nitrate (Chap. Ill), (2) after oxida-
tion to nitrate, conversion to molecular Ng ^^^ nitrous
oxide (Chap. Ill), (3) incorporation into organic molecules
(Chap. V). The anabolic aspects of nitrogen metabolism
culminate in the formation of two major groups of complex
substances, proteins and nucleic acids. The latter are con-
sidered in a separate chapter whilst protein synthesis is
traced step by step, beginning with the mode of incorpora-
tion of nitrogen from molecular Ng and NH3 into organic
molecules. After dealing with the synthesis of amino-acids
and with the mechanisms operative in the absorption of
these compounds from the environment, attention is next
directed to the significance of peptides in intermediary meta-
bolism, the problems of protein synthesis and how amino-
acids become joined together by peptide bonds. This part
of the monograph concludes with a chapter devoted to the
enzymes responsible for proteolysis, a process which ulti-
mately yields free amino-acids. The catabolism of the latter
is discussed at the beginning of the monograph, conse-
quently it will be appreciated that the metabolism of amino-
acids and proteins has been studied at various stages in a
cycle. The underlying theme of the monograph is none
other than that known to all biologists as the nitrogen
cycle, and an attempt has here been made to analyse some
of the component steps of the cycle from the standpoint of
the biochemistry of the various reactions and the micro-
organisms concerned (Fig. i.i).

Purely for convenience, and in order to avoid possible
confusion, the microbial metabolism of nucleotides, nucleo-
sides, purines and pyrimidines is discussed in a separate
chapter. This field is now being studied intensively and there
has been little time to correlate many of the experimental
facts rapidly being placed at our disposal. Partly for this
reason, and partly because of limitations in the amount of

CATABOLISM

ANABOLISM

denitrification

NO"

NO3 (ui)

nitrogen \

(Tv) NHjOH

fixation ^

PEPTIDES'*

FIG. I.I. — The Nitrogen Cycle. The roman numbers enclosed
within circles denote the numbers of the chapters dealing
with the various topics shown in the schenje

8 NITROGEN METABOLISM

Space available, the subject-matter of the chapter is con-
fined to a few selected topics.

In the concluding chapter the mode of action of chemo-
therapeutic agents is considered in terms of their observed
effects on the metabolism of compounds containing nitrogen.

BIBLIOGRAPHY

The following books are recommended for general reading and
as reference books for detailed information concerning specific
topics:

FOSTER, J. w. (1949), Chemical Activities of Fungi, Academic Press,
U.S.A.

FRY, B. A. and PEEL, J. L. (editors), (1954), Autotrophic Micro-
organisms, Soc. gen. Microbiol. Symp., 4, Cambridge Univer-
sity Press, G.B.

GALE, E. F. (1949), Chemical Activities of Bacteria, University
Tutorial Press, G.B.

LWOFF, A. ( 1 95 1 ), Biochemistry and Physiology of Protozoa, Academic
Press, U.S.A.

STEPHENSON, M. (1949), Bacterial Metabolism, Longmans Green,
G.B.

SUMNER, J. B. and myrback, k. (editors), (1950), The Enzymes,
Academic Press, U.S.A.

WERKMAn, c. H. and wilson, p. w. (editors), (195 1), Bacterial
Physiology, Academic Press, U.S.A.

REFERENCES

1. Albaum, H. G., Ogur, M. and Hirshfeld, A. (1950), Arch.

Biochem., 27, 130

2. Barker, H. A. (1950), in Phosphorus Metabolism, i, 204 (Ed.

McElroy, W. D. and Glass, B., Johns Hopkins Press,
U.S.A.)

3. and Lipmann, F. (1949), J. biol. Che?n., 179, 247

4. Dounce, A. L., Rothstein, A., Beyer, G. T., Meier, R. and

Freer, R. M. (1948), J. biol. Chem., 174, 361

5. Gest, H. (195 1), Bact. Rev., 15, 183

6. Hearon, J. Z. (195 1), Fed. Proc, 10, 602

7. Hersey, D. F. and Ajl, S. J. (1951), J. biol. Chem., 191, 113

8. Kaplan, N. O. in The Enzymes, 2 (i), Chap. 45

9. LePage, G. A. and Umbreit, W. W. (1943),^. biol. Chem., 147,

263; 148, 255

10. Lipmann, F. (1941), Advances in Enzymology, i, 99; (1946), 6,

231; (1949), Fed. Proc, 8, 597

11. Lohmann, K. (1928), Biochem. Z., 203, 164

INTRODUCTION

12.
13-

14-
15-

i6.

17-
i8.
19.

Lutwak-Mann, C. (1936), Biochem. J., 30, 1405
Mesrobeaunu, L. (1936), Thesis: Paris, Contribution a Vetude

des corps puriques de la cellule bacterienne
Michaelis, L. in The Enzymes, 2 (i), Chap. 44
O'Kane, D. J. and Umbreit, W. W. (1942). J. biol. Chem.,

142, 25
Racker, E. and Krimsky, I. (1952), Nature, 169, 1043
Vishniac, W. and Ochoa, S. (1952), J^. biol. Chem., 195, 75
Umbreit, W. W. (1947), Bact. Rev., 11, 157
Bacterial Physiology, Chaps. 11 and 19

CHAPTER II

AMINO-ACID CATABOLISM

Many heterotrophs can utilize organic nitrogen compounds,
in addition to carbohydrates, as primary sources of carbon
and energy. In general, the nitrogen is first removed from
the compound and the product is then fermented or oxidized
by the same terminal pathways that are operative in the
catabolism of carbohydrates and fatty acids. Certain hetero-
trophs, apparently lacking the ability to metabolize exogen-
ous sugars, are entirely dependent on organic nitrogen
compounds, such as amino-acids, purines or pyrimidines,
as sources of carbon and energy. Although the end-products
of the catabolism of these organisms have been studied,
little is yet known about the routes of their formation or the
enzymes responsible for the individual steps.

The catabolism of amino-acids commences either with an
oxidative deamination or with the removal of a specific
group by a non-oxidative process. It is unlikely that the
latter is directly responsible for making energy available to
the organism, but in either case examples are known in
which the further metabolism of the products proceeds by
routes which result in the formation of energy-rich phos-
phate groups. Thus pyruvate may arise by the non-oxida-
tive deamination of serine (p. 23) or the oxidative deamina-
tion of alanine (p. 11), and its oxidation by the pyruvic
oxidase system is accompanied by the formation of energy-
rich phosphate groups [39]. The first part of this chapter
is concerned with mechanisms and enzymes which accom-
plish the oxidative catabolism of amino-acids, whilst the
second part is devoted to enzyme systems whose primary
mode of attack is non-oxidative.

Amino-acid oxidases

The amino-acid oxidases oxidize amino-acids to the
corresponding keto acids and are specific for either the L or

10

AMINO-ACID CATABOLISM U

the D stereo-isomers of their substrates,

RCH(NH,)COOH+H20=RCOCOOH+NH3+2H

The transfer of hydrogen from the amino-acid to a suitable
acceptor, typically Og , appears to be mediated by one or
more carrier substances, and usually the enzyme has a
prosthetic group capable of functioning in this manner.
Enzymes of this type are the L-amino-acid oxidases of
Neurospora crassa and N. sitophila [7], Proteus vulgaris [58],
Penicillium notatum and Aspergillus niger [37]. Each of these
oxidases attacks a wide variety of amino-acids, although the
possibility that the observed activity is due to several very
similar, but specific, enzymes has not been ruled out.
Oxygen can be replaced in vitro by reducible dyes, such
as methylene blue, or by ferricyanide. There is evidence
that the enzyme from N. crassa possesses a prosthetic
group, adenine flavindinucleotide, which enables hydrogen
to be transferred directly to Og , resulting in the formation
of H2O2 [10]. In the presence of catalase (present in Neuro-
spora), the oxidation of one gram mole of amino-acid
involves the overall uptake of one gram atom of oxygen. The
mycelium of N. crassa also contains a similar oxidase
specific for D-amino-acids [7].

Whether the oxidase from Pr. vulgaris also has a flavin
prosthetic group has not yet been established, and although
one atom of oxygen is taken up per molecule of amino-acid
oxidized, there is no evidence that HgOg is first formed and
subsequently decomposed by catalase. There must be more
than one oxidase in Pr. vulgaris since washed suspensions
oxidize more amino-acids than the cell-free enzyme pre-
paration [58]. Oxygen is required for the deamination
of glycine, alanine and glutamic acid by washed cell sus-
pensions of Escherichia coli, Pseudomonas fluorescens and
Bacillus mycoides [cf. 25]: using cells treated with toluene to
prevent the further metabolism of pyruvate, it can be shown
that the deamination of alanine by Esch. coli proceeds
quantitatively according to the following equation:

CH3CH(NH2)COOH+i02=CH3COCOOH+NH3

12 NITROGEN METABOLISM

but nothing is known about the properties of the enzyme
concerned.

Glutamic acid dehydrogenase

The deamination of glutamic acid by Esch. coli is due to
the enzyme L-glutamic acid dehydrogenase with the co-
enzyme triphosphopyridine nucleotide (TPN) [2]: the end-
product is a-ketoglutaric acid and it is believed that the
reduction takes place in two stages:

COOH.(CH2)2CH(NH2)COOH+TPN+ ^

COOH.(CH2)2C(:NH)COOH+TPN.H+H+

COOH.(CH2)2C(:NH)COOH+H20 ^

COOH.(CH2)2COCOOH+NH3

The system is reversible, with the equilibrium in favour of
the synthesis of glutamic acid. A similar specific glutamic
dehydrogenase occurs in Saccharomyces cerevisiae [i],
Clostridium sporogenes [44], N. crassa [23], and probably
in Haemophilus pertussis [34] and H. parainfluenzae [36].
Haemophilus influenzae will not grow except in the presence
of a porphyrin (the X-factor) and diphosphopyridine
nucleotide (DPN), TPN or nicotinamide riboside (the
V- factor). The oxidative activity of cells harvested from a
medium deficient in the V-factor was considerably increased
by the addition of DPN or TPN, and in this way Klein
has shown that the latter are involved in the oxidation
of aspartic and glutamic acids to CO 2 , NH3 and acetic
acid [36]. Unlike H. parainfluenzae, no volatile fatty acid
was formed during the oxidation of amino-acids by H.
pertussis, an organism which requires neither X nor V fac-
tors, and in the experimental conditions employed by Jebb
and Tomlinson, only carbon from glutamic acid was incor-
porated into cell substance [34].

Oxidation of tryptophan by Pseudomonas spp.

A characteristic feature of species of Pseudomonas is that
they possess or quickly acquire the ability to utilize any one
of a wide variety of oxidizable organic substances as sources

AMINO-ACID CATABOLISM I3

of carbon and energy, a property which has been widely
exploited by Stanier in the elucidation of metabolic path-
ways by the technique of 'simultaneous adaptation' [54]. If
an organism exhibits little or no detectable activity against
a certain substance, and if the inclusion of this substance in
its environment evokes, in the absence of cell division, a
marked increase in the organism's ability to metabolize that
substance, then adaptation is said to have taken place. If an
organism can metabolize a particular compound, the hypo-
thesis of simultaneous adaptation postulates that it can also
metabolize immediately, and at a comparable rate, any sub-
stance which is an intermediate in the metabolism of that
compound (assuming that the intermediate can pass into the
cells). If there is a lag period prior to the rates of utilization
becoming comparable, then it may be concluded that the
substance cannot be attacked by the existing metabolic
systems, in other words, it is not an intermediate, and is only
metabolized after adaptation has taken place. From such
data it may be possible to deduce the probable route by
which a substance is catabolized, but unequivocal proof
requires not only direct evidence of formation of the inter-
mediates but also isolation of the appropriate enzymes. The
aerobic nature of the Pseudomonas spp. means that the
overall catabolism of whole cells can be studied mano-
metrically in terms of an uptake of O2 , and an example of
this technique is provided by the investigations concerned
with the degradation of tryptophan [55]. After being grown
on, or otherwise adapted to tryptophan, some strains of
Pseudomonas are simultaneously adapted to formylkynure-
nine, kynurenine, anthranilic acid and catechol; whilst
others are adapted to kynurenine and kynurenic acid, but
not to anthranilic acid or catechol. Work with cell-free
extracts [30] revealed that the pyrrole ring of tryptophan
(Fig. 2.1) is first ruptured by a peroxidase-oxidase system
in which both H2O2 and Og are involved, and the product,
formylkynurenine, is then hydrolysed by formylase into
formic acid and kynurenine. In some strains, the pyrrole
ring is now reformed, thus producing kynurenic acid, but
the route by which this substance is metabolized remains

14

NITROGEN METABOLISM

unknown. In other strains, kynurenine is split by kynuren-
inase into alanine and anthranilic acid. The oxidation of the
latter to COg proceeds via catechol, m-m-muconic acid,
and ^-ketoadipic acid. Whilst the three enzyme systems,
tryptophan peroxidase, kynureninase and pyrocatechase,
were all highly active in cell-free extracts from organisms

tryptophan
/^\ j|CH2Ch(nH2)cOOH

formyl
kynurenine

COOH

CH.COOH

I
CH.CH

II
HOOC.CH

muconic acid

HOOC.CH2CO(CH2)2COOH

/g-ketoadipic acid

FIG. 2.1. — Pathways of tryptophan degradation in
Pseudomonas spp,

grown in the presence of tryptophan, extracts from cells
grown on asparagine exhibited negligible activity. It is
notable that in none of the oxidative reactions could dyes
like methylene blue replace O2 as the H-acceptor. The
degradation of tryptophan via catechol has been termed the
'aromatic pathway' whilst that by way of kynurenic acid is
known as the 'quinoline pathway'. Animal tissues degrade
tryptophan by the former route. The routes of tryptophan

AMINO-ACID CATABOLISM 15

catabolism by Bacillus subtilis, another aerobe, appear to be
similar to those found in the Pseudomonas spp. [40].

Tryptophanase

The appearance of indole in the culture medium is a
valuable diagnostic test in bacteriology and it is readily
detected by the formation of a pink compound in the
presence of ^-dimethylaminobenzaldehyde and acid. Hop-
kins and Cole were the first to isolate tryptophan and imme-
diately suspected and proved that it was the natural
precursor of this indole. The enzyme system concerned has
been termed tryptophanase, and is found in some species of
Escherichia, Proteus and Vibrio, though there are strain
differences in any one species. Oxygen is probably not
directly involved in the initial step of the tryptophanase
reaction, but indole tends to accumulate only in aerobic
conditions; e.g. in the absence of Og , Esch. coli formed
indolepropionic acid and little, if any, indole. When washed
cells of Esch. coli were incubated with tryptophan and the
system adequately aerated, the uptake of oxygen corres-
ponded to that required for the complete oxidation of the
alanine side chain [64]. Most of the experiments concerned
with the mode of action of tryptophanase have been per-
formed with preparations of Esch. coli, and prior to attempt-
ing to prepare the enzyme system in a cell-free state atten-
tion was directed to the factors affecting its activity in whole
cells [29]. Tryptophanase was found to be adaptive, and
extremely active cells were obtained from vigorously aerated
media containing tryptophan. Whether Esch. coli grown in
the absence of the substrate exhibits detectable trypto-
phanase activity appears to depend on the strain of the
organism concerned. The inclusion of glucose in the
medium may result in the suppression of indole formation,
and if it does, the cells from such cultures do not exhibit
tryptophanase activity. This effect is not due to growth in
an acidic medium since other sugars are metabolized with
the formation of acidic end-products and yet they do not
suppress the adaptive formation of tryptophanase. How-
ever, several workers have reported activity in cells derived

l6 NITROGEN METABOLISM

from cultures grown in the presence of glucose and a
probable explanation of their results is that tryptophanase is
only developed after all or most of the glucose has been
decomposed. There is evidence that this enzyme system is
not developed in cultures grown in an amino-acid rich
medium containing glucose because phenylalanine and
tyrosine exert an inhibitory effect. The specificity of trypto-
phanase is high and indole is only formed from compounds
related to L-tryptophan provided that the a-carboxyl and
a-amino groups, the /^-position in the side chain, and the
N of the indole ring are unsubstituted.

Wood, Gunsalus and Umbreit [63] have obtained from
Esch. coll a cell-free preparation which attacked tryptophan
with the formation of equimolecular amounts of indole,
NH3 and pyruvic acid. Their tryptophanase preparation was
activated by pyridoxal phosphate and would not deaminate
either serine or alanine. They therefore concluded that
neither of these amino-acids is an intermediate in the
degradation of tryptophan by this route. Dawes and Hap-
pold have performed similar experiments and reached the
same conclusion. Although their system produced equi-
molecular amounts of indole and pyruvic acid there was an
excess of NH3 . Whilst no correlation with tryptophan dis-
appearance was attempted, these observations may indicate
that the initial step is one of deamination which is perhaps
catalysed by a type of L-amino-acid oxidase. If this were so,
the formation of ^-indolepyruvic acid would simultaneously
make hydrogen available for the reductive rupture of the
bond linking indole to the beta carbon of pyruvic acid:

i/^. nCH2CH(NH2)COOH

+ H0O, -NH3, -2H

NH

nCH,COCOOH

/\ . CH3

T^ Ml + CO

\A/ I

NH NH COOH

There is little direct evidence to support this hypothesis,

AMINO-ACID CATABOLISM I7

although it was noted that in addition to pyridoxal phos-
phate, the enzyme preparations contained riboflavin, a pos-
sible carrier of hydrogen. The bond joining indole to the
side chain appears to be susceptible to reduction since indole
is readily formed in vitro when tryptophan is either refluxed
with Raney nickel and absolute alcohol containing a little
HCl, or boiled with aqueous NaOH and catalytic amounts of
Cu"^"^ or Co"^"*". A summary of the other mechanisms which
have been proposed to explain the mode of action of trypto-
phanase will be found in the review by Happold [29].

With the possible exception of tryptophanase, all the
enzyme systems discussed above accomplish the oxidative
catabolism of amino-acids in association with molecular O2
as the ultimate and natural H-acceptor. In anaerobic organ-
isms either amino-acids themselves or compounds derived
from them may fulfil this function.

Stickland reaction

Stickland was the first to demonstrate that amino-acids
take part in anaerobic oxido-reduction reactions, certain
acids acting as H-donors whilst others function as Pi-
acceptors. The original experiments were done with the
strict anaerobe CI. sporogenes which is capable of growing
in an amino-acid medium in the absence of carbohydrates,
and he suggested that the organism derived its energy from
reactions of this type [56]. Nutritional studies later revealed
that the organism only grew well in a medium containing
adequate amounts of the amino-acids shown by Stickland
to be H-acceptors and H-donors [22]. In Stickland's experi-
ments, washed cell suspensions were incubated anaerobi-
cally with the appropriate substrates in Thunberg tubes.
Hydrogen-donor amino-acids were detected by their ability
to reduce methylene blue or cresyl blue to the colourless
leuco form. Alanine, valine, leucine and pyruvate were all
active H-donors whilst phenylalanine, aspartic and glutamic
acids showed some activity, reduction of the dye being
accompanied by deamination of the amino-acid. Hydrogen
acceptors were detected by their ability to accept hydrogen
from the leuco form of a dye of suitable redox potential

l8 NITROGEN METABOLISM

(e.g. phenosafranine and benzyl viologen, but not methy-
lene blue) and thus restore the original dye colour. Proline,
hydroxyproline and glycine were reduced by such a system,
though ammonia was only formed from glycine. Incubation
of alanine with proline resulted in the production of i mole
of NH3 per mole of alanine: none was produced from the
separate amino-acids. Stickland thus demonstrated that CI.
sporogenes catalysed oxido-reduction reactions between pairs
of amino-acids, one acid acting as a H-donor, the other as
a H-acceptor. The reduction of proline resulted in opening
of the ring and the formation of (5-aminovaleric acid, whilst
the products derived from alanine were NH3 , CO 2 and
acetic acid. When cresyl blue accepted hydrogen from
alanine, 2 moles of dye were decolorized for each mole of
NH3 released, indicating that the overall oxidation of
alanine involved the donation of four hydrogen atoms.
Although there was no direct evidence, it seemed highly
probable that pyruvate was an intermediate in the decompo-
sition of alanine and Stickland therefore proposed that the
overall reaction {d) represented the sum of three separate
reactions (a, b, c):

CH3CH(NH2)COOH+H.30=NH3+CH3COCOOH+2H (a)
CH3COCOOH+H20=CH3COOH+C02+2H (b)

4H+2NH(CH2)3CHCOOH=2NH2(CH2)4COOH (c)

CH3CH(NH2)COOH+2NH(CH2)3CHCOOH+2H20

=2NH,(CHo)4COOH+NH3+C02+CH3COOH (d)

similarly:

CH3CH(NH2)COOH+2NHoCH,COOH+2H20

=3NH3+C02+3CH3COOH

Clostridium botiilinum [15] and all the proteolytic Clos-
tridia examined by Nisman, Raynaud and Cohen [45] were
capable of performing the Stickland reaction; amongst the
organisms which could not were CI. tetani, CI. tetano-
morphum, CI. welchii and CI. saccharobutyricum. Apart from
the substances already mentioned, histidine, serine, iso-

AMINO-ACID CATABOLISM I9

leucine, tyrosine, methionine, ornithine, tryptophan,
phenylalanine, cysteine and ethanol also act as H-donors,
whilst tryptophan, tyrosine, ornithine and arginine function
as H-acceptors [33, 65]. It will be noted that some amino-
acids serve both as an acceptor and as a donor, and whilst
the reaction is specific for the L-isomer of H-donors [cf. 56],
there is no stereochemical specificity w^ith respect to
H-acceptors [65].

If CI. sporogenes is grown in the presence of glucose, it
develops an active hydrogenase which enables the reducing
component of the Stickland system to be replaced by mole-
cular H2 [33]. The reaction can then be followed in terms
of an uptake of Hg and the end-products are only those
derived from the amino-acid added as the H-acceptor. In
an analogous manner, and perhaps unexpectedly since the
organisms are strict anaerobes, the H-acceptor part of the
system can be replaced by Og [46]. A number of amino-
acids, all H-donors in the Stickland reaction, were oxida-
tively deaminated by washed suspensions of CI. sporogenes
to the corresponding a-keto acid. The uptake of Og was
appreciably reduced by the presence of a H-acceptor amino-
acid and restored to its former value by the addition of
arsenite. The latter can readily be explained since although
arsenite completely inhibited H-transfer to a H-acceptor
amino-acid (e.g. from leucophenosafranine to proline) it
had no eflFect on the H-donor part of the system (e.g. the
reduction of phenosafranine by alanine) [47]. Hence the first
step in the Stickland reaction is probably catalysed by a
type of L-amino-acid oxidase which, perhaps with the aid
of one or more carriers, can transfer hydrogen to Og, a dye
or another amino-acid. In cell-free extracts, DPN but not
TPN was readily reduced by H-donor acids [44], but
whether reduced DPN is the natural carrier and reacts
directly with H-acceptor amino-acids is not known. Pyru-
vate and other a-keto acids are oxidized by CI. sporogenes to
CO 2 and a fatty acid containing one less carbon atom by a
mechanism which can lead to the fornjation of energy-
rich phosphate groups (cf. reaction h):

CH3COCOOH+H3PO4— > CHgCO^POsHa+zH+CO., [47]

20 NITROGEN METABOLISM

Nisman and his colleagues therefore support Stickland's
concept of the reaction mechanism and they believe that
reactions «, h and c are catalysed by an L-amino-acid
oxidase [51], a keto acid oxidase [cf. 39] and an amino-acid
reductase respectively. There is a complete lack of know-
ledge concerning the mechanism by which an amino-acid is
reduced to a fatty acid (reaction c). Reaction a is inhibited
by KCN and secondary octyl alcohol, b by iodoacetate and
c by arsenite [47]. The growth of CI. sporogenes at the ex-
pense of energy derived from amino-acids is characterized
by the production of acetic acid together with isobutyric,
isovaleric and optically active valeric acids, derived respec-
tively from valine, leucine and isoleucine by the Stickland
reaction. On the other hand, owing to its inhibitory effect
on the Stickland reaction, the utilization of glucose as the
energy source yields only acetic and butyric acids [16].

Fermentation of amino-acids by other organisms [20]

Certain organisms live anaerobically by the fermentation
of one particular organic nitrogen compound whilst others,
although not so specific, are restricted to the utilization of
a small number of chemically related compounds. These
organisms have usually been isolated by the enrichment
culture technique and the anaerobic incubation of a sample
of mud or soil in a medium containing an organic nitrogen
compound as the major source of carbon. In the event of the
organisms requiring certain growth factors, a small amount
of yeast extract is usually included in the medium after the
first transfer.

The anaerobic cocci Diplococcus glycinophilus [11], Micro-
coccus anaerobius and M. variabilis [19] are specific for
glycine, and no other substance is readily metabolized unless
glycine is also present. If cultures of D. glycinophilus are not
shaken, the overall fermentation is expressed by:

4NH2CH2COOH+2H20=4NH3+3CH3COOH+2COo

The fermentation is not a simple dismutation involving the
oxidation of one molecule of glycine to CO 2 and NH3 and
the reduction of three molecules to acetic acid and NH3.

AMINO-ACID CATABOLISM 21

Experiments with isotopically labelled glycine have shown
that most of the methylene carbon appeared as acetic acid
whilst the carboxyl carbon appeared as CO 2 [4].

Alanine, serine or threonine serve as sole sources of carbon
and energy for CI. propionicum and are fermented to CO 2 ,
NH3 and fatty acids [11, 12]:
3CH3CH(NH2)COOH+2HoO =

2CH3CH,COOH+CH3COOH+CO,+3NH3
3CHo(OH)CH(NH.,)COOH+H.,0 =

CH3CH2C6OH+2CH3COOH+2CO0+3NH3
The mechanism of alanine fermentation may be comparable
with that proposed for the Stickland reaction, alanine acting
both as the H-acceptor and as the H-donor. Lactate and
pyruvate are fermented in a similar manner and these fer-
mentations are at least superficially comparable to the fer-
mentation of lactate by the propionibacteria. However,
whilst in the latter propionic acid arises by the decarboxyla-
tion of succinate, in CI. propionicum it is probably formed
from acrylate [35]. Barker and Wiken have concluded that
acetate is not an intermediate in the fermentation of threo-
nine to butyric and propionic acids, and that butyric acid
probably arises directly from a C4-compound (a-ketobu-
tyrate?)[5].

Unlike D. glycinophilus and CI. propionicum, CI. tetano-

morphum ferments glucose as well as certain amino-acids.

The end-products of both glutamic acid and histidine

fermentations include Hg , CO2 , NH3 , acetic and butyric

acids [66], and by analogy with Edlbacher's work with liver,

Woods and Clifton were the first to suggest that glutamic

acid was an intermediate in the fermentation of histidine.

Confirmation of this hypothesis has been recently obtained

and the first step in the conversion of histidine (I) to

glutamic acid, HCOOH and NH3 involves deamination to

urocanic acid (II) [60].

CH=CH.CH2CH(NH2)COOH

h ^i >

N3 iNH -NH3

CH

(I)

22 NITROGEN METABOLISM

CH=CH.CH:CHCOOH (CH2)2COOH

II I 1

N NH — >CH(NH2) +HCOOH+NH3

\ / I

CH COOH

(11)
Urocanic acid and glutamic acid are also intermediates in
the oxidation of histidine by Ps. fluorescens, and isotopes
have been used to show that the amino-nitrogen of glutamic
acid and the carbon of HCOOH are derived from the N and
C in positions i and 2 of the imidazole ring [59]. The fer-
mentations of C/. tetani [48Z>]and CI. cochlearum [3] resemble
those of CI. tetanomorphum. The former ferments a number
of amino-acids, only one of which, histidine, is among those
essential for growth; aspartic acid and serine give rise to
alcohols as well as to fatty acids. It is well known that yeasts
fermenting carbohydrates in the presence of amino-acids
produce a number of the higher aliphatic alcohols (fusel oil),
and Ehrlich showed that the latter contain one less carbon
atom than the amino-acids from which they were derived,
the overall reaction being:

Such a process may be the means whereby the nitrogen of
the amino-acid is made available in the form of NH3 , but
the details of the mechanism are unknown. Most of the
alcohols are formed after the amino-acids have disappeared
from the medium, indicating that there are intermedi-
ate stages between deamination and production of the
alcohol [13].

Anaerobic ac- deaminases

A characteristic feature of the anaerobic a- deaminases is
that the reaction product is unsaturated and therefore such
enzymes are sometimes described as desaturases. One
typical example has already been mentioned, namely, the
enzyme forming urocanic acid from histidine, and there is
evidence that it also occurs in certain strains of Esch. colt,
Salmonella paratyphi and Shigella paradysenteriae [50]. A
similar type of reaction is catalysed by aspartase, an enzyme

AMINO-ACID CATABOLISM 23

found in Esch. colt, Ps. fluorescens, Serratia marcescens, Pr.
vulgaris, Lb. casei, and perhaps in yeasts [67, 17, cf. 21]:

COOH.CH2CH(NH2)COOH # COOH.CHrCH.COOH+NHs

The system is reversible and by using cells treated with
cyclohexanol to prevent conversion of the fumarate to suc-
cinate or malate, it can be shown that the equilibrium
favours the synthesis of L-aspartic acid [67]. Serine and
threonine are deaminated anaerobically to pyruvic and
a-ketobutyric acid respectively, though whether one de-
aminase catalyses both reactions is not yet known. Unlike
aspartase, the reaction does not appear to be reversible and
the first step is thought to be the removal of the elements
of water, followed by the spontaneous hydrolysis of the
resulting imino-compound [i4<2]:

CH2(OH)CH(NH2)COOH > (CH2:C(NH2)COOH) -^

CH3C(:NH)COOH

CH3C(:NH)COOH ^ CH3COCOOH+NH3

The dehydration step is analogous to that catalysed by
enolase, and cysteine desulphurase may likewise be regarded
as first removing the elements of HgS:

CH2(SH)CH(NH2)COOH+H20=CH3COCOOH+H2S+NH3

Serine and threonine deaminase activity is found in Esch.
colt, CI. welchii, Ps. pyocyanea, Proteus OX-ig and staphylo-
cocci: cysteine desulphurase occurs in Sac. cerevisiae, Esch.
colt, Pr. vulgaris, B. subtilis and Propionibacterium pentosea-
ceum [24].

Aspartase, cysteine desulphurase, and the serine and
threonine deaminases are all alike in that their activity is
dependent on the presence of certain co-factors, the identity
of which has not yet been completely established. For
example, there was a marked reduction in the aspartase
activity of washed cell suspensions of Esch. coli after they
had been kept standing in water or buffer. This decay in
activity could be prevented by the addition of a small amount
of either adenylic acid (AMP) or orthophosphate, together
3

24 NITROGEN METABOLISM

with a reducing agent such as cysteine, lactate or for-
mate [28, 25]. Similarly, a loss of serine deaminase activity
was prevented by glutathione, formate or AMP. During
investigations of the nutrition of lactobacilli and Strep,
faecalisy organisms exacting towards aspartic acid, Stokes,
Larsen and Gunness observed that they grew well in the
absence of this amino-acid provided the medium contained
adequate amounts of biotin. They therefore suggested that
biotin plays an importarit role in aspartic acid metabolism,
and perhaps especially in the aspartase system, although
they were unable to demonstrate the presence of the latter
in these organisms [57].

Meanwhile Lichstein and his colleagues were studying
the catabolism of aspartic acid and serine by Esch. coli, Pr.
vulgaris and Bacterium cadaveris. They found that of the
known B vitamins only biotin was capable of reactivating
washed cell suspensions whose deaminase activity had been
reduced by standing for 30 minutes in molar phosphate
buffer, pH 4 [42]. This loss of deaminase activity and subse-
quent reactivation by biotin or by much larger concentra-
tions of AMP was only exhibited by cells which had been
grown in the presence of a yeast extract. A concentrate of
compounds containing biotin was prepared from yeast and
was found to be a hundred times more effective in enhancing
the aspartase activity of 'aged' suspensions than was to be
expected on the basis of its biotin content. Moreover, when
fresh, a partially resolved cell-free preparation of aspartase
was activated either by yeast extract or by biotin plus
adenylic acid, but after standing at 0° C, only the former
was effective [41]. A bound form of biotin, termed biocytin,
has been isolated recently from yeast, crystallized and shown
to be £-N-biotinyl-L-lysine [69]. Biocytin itself is probably
not the natural coenzyme, since, unlike the impure concen-
trates, its activity is only comparable to that produced
by an equivalent amount of free biotin [68]. Wood and
Gunsalus have prepared from Esch. coli a purified cell-free
system possessing serine and threonine deaminase activity,
and although both adenylic acid and glutathione were
required, biotin did not appear to be necessary [62\

AMINO-ACID CATABOLISM 25

Furthermore, Binkley's dialysed cell-free preparation of
serine deaminase was reactivated by Zn [8]. In contrast
with this work with bacteria, it is now clear that Neurospora
possesses two deaminases — one specific for the L isomers of
threonine and serine [71], the other for the D isomers [70]
— and that both of these enzymes are activated by pyridoxal
phosphate. Biotin, AMP and GSH, either separately or
together, did not affect the activity of cell-free prepara-
tions. Though the D-serine deaminase of Esch. coli is like-
wise activated by pyridoxal phosphate, attempts to show
that the corresponding L-serine deaminase is activated by
this substance have been unsuccessful [43^]. Pyridoxal
phosphate is also reported to be the activator of the cysteine
desulphurase of Proteus vulgaris [48^]. In view of these
diverse observations, it is not yet possible to define the co-
factors naturally associated with the anaerobic deaminases.
The confusion in this field has recently been increased since
a substance produced by heating glucose with acid under
pressure was found to be as active as yeast extract in the
activation of the resolved aspartase system of Bact. cadaveris
and yet did not contain biotin [146]. Winzler, Burk and du
Vigneaud [61] have observed that unless biotin was added to
the system, washed cells of biotin-deficient Sac. cerevisiae
were incapable of assimilating exogenous NH|. Their ob-
servation can be readily explained, if, as Lichstein's work
indicates, this growth factor is an essential component of
aspartase, one of the systems by which inorganic nitrogen is
incorporated into organic molecules (p. 60).

In general, the anaerobic deaminases are most active in
cells harvested from cultures at the cessation of active
cell-division. Moreover, the presence of Og favours the
development of the aerobic amino-acid oxidases whilst
anaerobic conditions favour the anaerobic deaminases. The
metabolic quotients of the former (QnHs about 30) are very
much lower than those of the latter (Qnh3=2oo-i,ooo).
After growth in the presence of glucose, organisms usually
possess poor deaminase activity, and although in general the
optimum pH for deaminase activity is in the range 8-10,
this effect cannot be explained in terms of growth in an

26 NITROGEN METABOLISM

acidic environment since activity is not increased by buffer-
ing the medium. Nor must it be assumed that glucose is
used preferentially as a source of carbon and energy and
that in consequence the organisms tend not to synthesize
the deaminases. On the contrary, the reduced activity may
be a reflection of the lack of essential co-factors since Boyd
and Lichstein [9] found that the low serine deaminase
activity of a washed suspension of Esch. coli (grown in the
presence of glucose) was almost immediately increased by
the addition of biotin, adenylic acid, yeast extract or liver
extract.

Having considered the enzymes which deaminate amino-
acids, it is now convenient to deal with those which attack
X)ther groups in the molecule.

Arginine dihydrolase

Washed suspensions of Strep, faecalis, Staph, aureus^
'CI. septicum and CI. sporogenes decompose arginine into
ornithine, NH3 and CO 2 :

C.NH(CH2)3CH(NH2)COOH+2H20

NH

=2NH3+C02+NH2(CH2)3CH(NH2)COOH

Since urea is not attacked by Strep, faecalis, the overall
reaction cannot be explained in terms of the splitting of
arginine into ornithine and urea by arginase and the subse-
quent decomposition of urea by urease. Hills [32] therefore
proposed that the enzyme system in Strep, faecalis and
Staph, aureus should be known as arginine dihydrolase.
Recent work has shown that more than one enzyme is in-
volved. The first step in the reaction is the formation of am-
monia and citrulline, NH2CONH(CH2)3CH(NH2)COOH,
which is then degraded to ornithine by an enzyme system
activated by inorganic phosphate, Mg"*""^ or Mn"^"*", and
ATP or AMP [38, 52]. Strep, faecalis is exacting towards
arginine, and arginine can be replaced by ornithine only if
the medium also contains adequate amounts of CO 2 . Carbon

AMINO-ACID CATABOLISM 27

dioxide is not produced during the fermentation of glucose
by Strep, faecalis, and since growth is not possible in its
absence Gale has suggested that arginine dihydrolase is a
mechanism whereby this metabolite is made available [26].
He found that strains with high arginine dihydrolase activity
grew better if the amount of arginine initially present in
the medium was such that it was not all decomposed by
the time the pH became unfavourable for further growth.
Alternatively, it is feasible that arginine serves as a source
of energy and that arginine dihydrolase activity is connected
with the organism's energy metabolism, since Knivett [38]
has shown that the conversion of citrulline to ornithine is
accompanied by the phosphorylation of ADP to ATP.

Amino-acid decarboxylases

Although for several years bacteria have been known to
form amines from amino-acids, no study of the enzymes
concerned was made until the work of Gale [27]. The amines
arise by decarboxylation of a-amino-acids:

RCH(NH2)COOH=RCH2NH2+C02

and the initial experiments demonstrated the existence of
six amino-acid decarboxylases, specific respectively for the
L-isomers of tyrosine, lysine, ornithine, arginine, histidine
and glutamic acid. They have been found in the genera
Escherichia, Streptococcus, Clostridium, Proteus and Lacto-
bacillus. Whilst an organism may possess more than one
decarboxylase, the distribution of the enzymes amongst the
strains of any one species is variable. These enzymes are
active in the pH range 2-6 and have a sharp optimum. In
general the substrates possess an a-amino and an a-carboxyl
group together with another polar group at the opposite end
of the molecule. Substitution in any of these groups yields
substances which are not attacked and the introduction of
a hydroxyl group in another part of the molecule is the only
known structural modification which does not affect suscep-
tibility to decarboxylation, e.g. ^-hydroxyglutamic acid,
/5-hydroxylysine and 3:4-dihydroxyphenylalanine are decar-
boxylated by the glutamic, lysine and tyrosine decarboxylases

28 NITROGEN METABOLISM

respectively. Because of their specificity and the fact that
decarboxylation proceeds to completion, these enzymes
are invaluable for the quantitative determination of the
corresponding amino-acids. The analytical procedure origi-
nated by Gale is based on the use of a washed suspension
or an acetone powder of the appropriate organism and the
manometric determination of the CO 2 released.

The growth conditions are especially important in deter-
mining whether the cells develop highly active decar-
boxylases, and owing to their adaptive nature, the medium
must contain the specific substrate. The enzymes are not
formed unless growth occurs in an acid environment, and
this is usually attained by allowing the organisms to meta-
bolize glucose. Temperature is also important: for example,
the decarboxylase activity of Esch. colt is better when cul-
tures are grown at 20-26° C. rather than at 37° C, but the
opposite is true of Strep, faecalis and CI. welchii. Decar-
boxylase activity becomes maximal at the cessation of active
cell division. Except for the histidine enzyme, there is
evidence that all the amino-acid decarboxylases possess a
prosthetic group, codecarboxylase, the existence of which
was first discovered by Gale during the purification of
the lysine decarboxylase. Fractionation with (NH4)2S04 in
alkaline conditions resulted in the precipitation of an in-
active apoenzyme to which decarboxylase activity could be
restored by the addition of extracts of bacteria, yeast or
animal tissues. Gunsalus and his co-workers later identified
codecarboxylase as being pyridoxal phosphate. It is there-
fore essential that the growth medium should contain ade-
quate amounts of pyridoxin, since even when the organism
is not exacting towards this factor, the rate of synthesis may
be insufficient to saturate the apoenzyme. The apoenzyme
of tyrosine decarboxylase can be prepared [6] by growing
Strep, faecalis in a pyridoxin-deficient medium containing
D-alanine, a substance which, according to the strain,
replaces or reduces the organism's pyridoxin requirements.
Washed cells of bacteria grown in this way exhibit no
activity unless the experimental system contains either
synthetic pyridoxal phosphate, pyridoxal or natural code-

AMINO-ACID CATABOLISM 29

carboxylase, and they can therefore be used for assaying
codecarboxylase .

More recent work has shown that the tyrosine de-
carboxylase will also attack phenylalanine [43^2] and m-
tyrosine [53], and that Esch. colt possesses a specific enzyme
which decarboxylates a,e-diaminopimelic acid to lysine.
Unlike the other decarboxylases, the activity of the latter is
high even in cells grown in the absence of the substrate, and
furthermore there is evidence that the reaction is reversible:
like the other decarboxylases the prosthetic group appears to
be pyridoxal phosphate [18]. Cultures of Proteus spp. grown
in an amino-acid medium have been found to contain amines
derived from the branched chain amino-acids valine, leucine
and isoleucine, but their mode of formation is not yet
known [49].

Fermentations usually give rise to acidic end-products,
consequently as the fermentation proceeds, the pH of the
medium eventually reaches a value below which no further
growth is possible. The highly basic amines formed as the
result of decarboxylase activity tend to counteract this fall
in pH, and Gale has therefore proposed that the decar-
boxylases may be regarded as a type of 'neutralization
mechanism' [27]. Since the partial pressure of CO 2 in an
acidic medium is low, another possible function of the
decarboxylases is to make this essential metabolite available
inside the cell. The observation that H. parainfluenzae is
exacting towards putrescine, the amine formed by the
decarboxylation of ornithine, implies that the amines them-
selves may be of some significance in intermediary meta-
bolism [31].

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16. Cohen-Bazire, G., Cohen, G. N. and Prevot, A. R. (1948),

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17. Cook, R. P. and Woolf, B. (1928), Biochem. J., 22, 474

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20. Elsden, S. R. (1952), in The Enzymes, 2 (ii), Chap. 65

21. Erkama, J. and Virtanen, A. I. (1951), The Enzymes, i (ii),

Chap. 42

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16, 326

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31. Herbst, E. J. and Snell, E. E. (1948), J. biol. Chem., 176, 989

32. Hills, G. M. (1940), Biochem. J., 34, 1057

33. Hoogerheide, J. G. and Kocholaty, W. (1938), Biochem. J.,

32, 949, 437

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Microbiol., 5, 951

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AMINO-ACID CATABOLISM 3I

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171

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71- and Reissig, J. L. (1953), J. biol. Chem., 202, 567

CHAPTER III

NITRIFICATION AND DENITRIFICATION

Nitrification

Nitrosomonas and Nitrohacter are two genera of strictly
aerobic chemosynthetic autotrophs which respectively
obtain their energy by the oxidation of ammonia to nitrite
(NOo), and nitrite to nitrate (NO3 ).

Nitrosomonas, NH3+iJ02=HN02+H20
Nitrohacter, N02+i02=N07

These organisms play an important role in the formation
of NO"^ from NH3 and organic nitrogen compounds, a pro-
cess occurring in soil and the beds of sewage purification
works [7] and known as nitrification. Most organic nitrogen
compounds are only nitrified by Nitrosomonas an^ Nitro-
hacter after they have been degraded by heterotrophs to

NH3.

The first evidence in favour of Pasteur's suggestion that
nitrification was due to micro-organisms came from the
classic experiments of Schloessing and Muntz [27]. After
sewage effluent had percolated through a column of sand
and chalk for about twenty days, they noted that NH3 was
being converted almost quantitatively into NO"^. This con-
version was completely stopped if the column was subjected
to conditions injurious to life, e.g. heat or chloroform, but
commenced again after washing with non-sterile water
derived from soil. Of the organisms known at that time,
none could oxidize NH3 to NO 7, and the first attempts to
isolate the causative agents by plating soil on nutrient
gelatin media all ended in complete failure. The lack of
success was traced to the inhibitory effect of organic sub-
stances on the growth of nitrifying organisms and resulted
in the introduction of media containing only inorganic

32

NITRIFICATION 33

salts [33]. After inoculating such media with soil, Wino-
gradsky readily obtained excellent growth accompanied by
nitrification, and the cultures were easily maintained
through successive transfers in a mineral medium containing
potassium phosphate, NH4CI, MgS04 and K2CO3 . Using
the enrichment culture technique, the isolation of organisms
oxidizing NH3 was facilitated by repeated subculture in an
inorganic medium containing NHt but no added NOT:
similarly, a medium containing N07 and no NH^ was used
for organisms oxidizing NOT. Winogradsky solved the
problem of how to obtain discrete colonies on a solid yet
completely inorganic medium by using silica gel as the
matrix for the mineral salts [34]. The colonies are minute
(diam. 200 [.i) and micro-manipulators are sometimes used
to pick out and transfer those required for inoculating sub-
cultures. Winogradsky was the first to obtain indisputably
pure cultures of organisms which specifically oxidized either
NH3 (Nttrosomonas europaed) or NOT {Nitrohacter wino-
gradsky). About the same time, Warington [31] and the
Franklands [8], working independently, obtained cultures
which oxidized NH3 to NOT and which appeared to be
pure as judged by microscopic examination and the absence
of growth on gelatin plates. The isolation of pure cultures
of the nitrifiers is difficult because their slow growth favours
the appearance of heterotrophs which grow rapidly even if
only traces of organic matter are present in the medium.
Great care is therefore required in assessing the purity of
any culture of nitrifying bacteria and no growth should be
detectable after incubating plates of nutrient agar streaked
with such cultures. Lees has found that adequate aeration
of the medium greatly facilitates the isolation and culturing
oi Nttrosomonas [14].

Some strains of the nitrifiers are actively motile whilst
others appear to be habitually associated with surfaces, e.g.
they readily adhere to granules of CaCOg [cf. 19]. Many of
these organisms prefer a slightly alkaline environment [27,
18], and for this reason CaCOg , K2CO3 or MgC03 are fre-
quently added to the medium [34, 20, 12]. Such substances

34 NITROGEN METABOLISM

serve as a source of COg and at the same time prevent the
H"^ produced during nitrification from lowering the pH to
a value unsuitable for growth [20]. The optimum pH for
the growth of a particular strain tends to be related to the
pH of the soil from which it was isolated, e.g. one from
a peaty soil will nitrify in relatively acid conditions [11].
Meyerhof observed that the growth of Nitrosomonas and
Nitrohacter is inhibited by high concentrations of their
respective substrates. Furthermore, high concentrations of
NHt also inhibited the oxidation of NO^, and the higher
the pH of the medium the greater the inhibition, both of
growth and of nitrite oxidation. Both NHt and NO^ in
excessive concentrations are known to be injurious to cells
in general, and the effect of pH may be explicable on the
basis that conditions of high pH favour the formation of
unionized NH3 which may enter the cells more easily than
the ammonium ion [20].

The autotrophic nature of the nitrifiers, together with
their apparent inability to grow in organic media, led Wino-
gradsky to conclude that they neither required nor utilized
organic nutrients. There has been much controversy as to
whether all organic materials are toxic to these organisms
and whether they can in fact assimilate at least some of these
substances. There is now a certain amount of evidence that
their growth is stimulated by small amounts of peptone, by
yeast extracts and by a partial hydrolysate of egg albumin [9]:
moreover, some strains of Nitrosomonas can grow in the
presence of high concentrations of glucose (10%) [11, cf. 12].
Some evidence that the nitrifiers may be able to obtain
energy in a manner typical of heterotrophs, namely by the
degradation of organic compounds, has been obtained by
Bomeke. In manometric experiments with thick suspen-
sions of Nitrosomonas and Nitrohacter y he found that in the
absence of exogenous nutrients, these organisms absorbed
O2 , indicating that they could obtain energy by the oxida-
tion of endogenous reserve materials [6]. Whilst there have
been many experiments purporting to show that organic
media support the growth of the nitrifiers, the majority of

NITRIFICATION

35

these investigations have been discounted on the grounds
that the cultures were contaminated with heterotrophs [35,

percolating fluid
drawn up tube
by suction

capillary
tube

to suction
pump «.

air or gas inlet
and sampling tube

FIG. 3.1. — Lees' soil percolation apparatus, as modified
by Audus [3]

12]. Quastel and Scholefield have pointed out that organic
inhibitors fall into two groups, those which inhibit in con-
centrations of 0-0 1 M. or less and those which inhibit only

36 NITROGEN METABOLISM

in very high concentrations [22]. But it must also be
emphasized that the inhibitory action of any substance is
determined by the organism concerned and also by the
physical properties of the environment, e.g. peptone is far
less inhibitory in the presence of sand than in ordinary
liquid cultures [32].

Owing to their slow growth and the difficulty of isolating
pure cultures, there have been few comprehensive investi-
gations of the metabolism of Nitrosomonas and Nitrohacter.
Meyerhof has dealt with the effect of substrate concentra-
tion, inorganic ions and various inhibitors on the rate of
nitrification in liquid cultures [20]. Nitrification under con-
trolled conditions in soil has been studied more recently by
Lees and Quastel using a technique based on the continuous
repercolation of fluid through a column of soil [16]. This
was accomplished by a simple apparatus which ensured that
the soil was nearly saturated with water and was adequately
aerated (cf. Fig. 3.1).

When NHt was added to the percolating fluid there was
a lag period after which the rate of nitrate formation
gradually increased and at the end of five days became
linear. During the first two days small quantities of NO "2
were detected. A graph of the progress of nitrification is
reminiscent of that expressing the rate of growth of a
bacterial culture and it appears that the soil ultimately
becomes saturated with nitrifying bacteria. At this stage the
behaviour of the system is analogous to that of a washed
cell suspension, i.e. it is metabolically active but the organ-
isms are not actively dividing. When a fresh solution con-
taining NH| was percolated through such a 'saturated soil',
nitrification occurred at a linear rate from the beginning
(Fig. 3.2). Consequently when the 'saturated soil' is treated
with a substance which is nitrified directly, NO 7 formation
should commence at once and at a maximal and linear rate.
A lag period implies that the substance can only be nitrified
after being converted into another compound and that time
is required for the formation of the appropriate adaptive
enzymes in the existing bacteria or for the growth of new

NITRIFICATION 37

organisms. For example, NO^ is immediately nitrified by
soil which has been previously percolated with NHt, but
there is a lag in the nitrification of NHt by soil previously
treated with NO^. Hence the 'saturated soil' technique is
of value in testing substances believed to be intermediates
in the nitrification process. Thus hydroxylamine (NHgOH)
has long been postulated as a possible intermediate in the
conversion of NH3 to HNO2 , but there is little direct

i20-| f^ r-l-9

FIG. 3.2. — Course of nitrification in soil percolated with o-oi M.-
NH4CI: A, first percolation of fresh soil; B and C, linear rela-
tionship between time and logio /xg. N07-N/ml. formed
respectively in the first (B) and second (C) percolations [22]

evidence [15]. Free NHgOH is toxic to bacteria and soon
stops nitrification. However, when combined, as in pyruvic
oxime, it is nitrified by soil enriched with nitrifiers, but
only after a lag. This is due to the development of hetero-
trophs, species of Archromohacter and Corynebacteria, which
convert the NHgOH into NOT without the intermediate
formation of NH3 [23]. The percolation technique is also
valuable for determining the effects of various substances
on nitrification under conditions simulating those found in

38 NITROGEN METABOLISM

nature. Most natural organic compounds, such as amino-
acids, are not inhibitory, methionine being a notable excep-
tion. Although potassium chlorate in low concentrations
(e.g. io~^ M.) prevents the proliferation of Nitrohacter, it
did not affect either the growth of Nitrosomonas or the
oxidation of NO 2^ by an established culture of Nitrohacter.
Chloromycetin, an antibiotic containing a nitro group, is
especially active against organisms oxidizing NO 2" [22].

The rate of nitrification was found to be a function of the
amount of NH 4 adsorbed by the base exchange complexes
in the soil and could be increased by increasing the soil's
base exchange capacity. No such effect was produced by the
addition of materials (e.g. sand) whose only effect was to
increase the available surface area. Moreover, the presence
of ions such as Ca++, which can displace NHt, depressed
the rate of nitrification. Few bacteria were found in the per-
colating fluid itself, and Lees and Quastel deduced that the
nitrifying organisms grow on the surface of soil particles
[cf. 16] around receptor areas which combine with or adsorb
NHt- All these areas are occupied in a 'saturated soil' and
further growth of the bacteria is restricted to replacing dead
cells [16]. Quastel and Scholefield have developed a tech-
nique whereby the Warburg manometer can be used in
studies of soil metabolism and they showed that following
the addition of NHt or NO 7 to soil saturated with nitri-
fying bacteria, the Og uptake was equal to that required for
complete oxidation to NO 7 [22].

Nothing is known about the mechanisms which enable
the organisms to utilize the energy made available by the
oxidation of NH3 and NO^. Ammonia is oxidized even
when the cells are unable to grow, e.g. in the absence of
CO 2 [21c], and the suggestion has been made that a metal-
activated enzyme may be involved in this process [15].
With regard to energy relationships and the efficiency of
energy utilization, the best data are those of Baas-Beck-
ing and Parks, who calculated the changes in free energy
(AF) taking place at 25° C. in conditions shown by Meyer-

DENITRIFICATION 39

hof [4] to be Optimal for nitrification (NH 4" =0-005 ^-J
H"^ = io-^ M.; N07=3-o3 m.).

NHt + ii02=N07+H20 + 2H+, AF=-66-5 kg. cal.
N02+i02=N03, AF=- 17-5 kg. cal.

The amount of carbon assimilated as the result of the oxida-
tion of a known amount of NH^ or NO 2^ is determined
experimentally, and assuming that the reduction of i gram
mole of CO 2 to CHgO (the generalized formula for cell sub-
stance) requires 118 kg. cal. of free energy, it can be calcu-
lated that the energy released in the oxidations is used with
an efficiency of S'9% by Nitrosomonas and 7*8% by Nitro-
bacter [4]. These values are only approximate, and in the
case of Nitrosomonas it is known that the efficiency falls as
the culture grows older [lo].

Reduction of nitrate: denitrification

In contrast to the limited number of organisms capable
of oxidizing ammonia and nitrite, several species accom-
plish the reverse process, namely, the reduction of nitrate and
nitrite to ammonia, nitrous oxide (N2O) and molecular
nitrogen. Whilst ammonia may be retained in the organism
or in the medium, gaseous products such as Ng and NgO
pass readily into the atmosphere with the result that the
overall nitrogen content of the organism's immediate
environment is decreased. In the latter instance the bio-
logical reduction of NO 7 and NOT is often termed de-
nitrification. Examples of organisms known to reduce
NO 7 include Ps. fluorescens, Ps. denitrificans, Ps. stutzeri^
Mia-ococcus denitrificans, various spore-forming bacilli,
Thiobacillus denitrificans, N. crassa, Hafisenula anonialay
Azotobacter agilis, Esch. coli and CI. welchii. The latter five
species appear to be incapable of taking the reduction as
far as NgO or Ng .

With some organisms, NO"^ and NO^ serve as sources

of nitrogen for the synthesis of organic nitrogen compounds:

alternatively, or in addition, they may function as H-

acceptors in reactions concerned with the organism's energy

4

40 NITROGEN METABOLISM

metabolism. Since it is generally believed that only inorganic
nitrogen in the forms of NH3 can be incorporated into
organic molecules, it is probable that in both cases the
metabolic pathways have at least the initial steps in common.
The end-result is different in that one leads to the assimila-
tion of nitrogen, whilst in the other the products are excreted
into the medium. The ability to reduce NO7 enables certain
organisms to grow anaerobically in media which would
otherwise only support their growth in the presence of O, ,
and in such cases NO 7 may be regarded as replacing oxygen
as the ultimate acceptor of metabolic hydrogen [25]. For
example, Esch. colt cannot grow anaerobically on lactic acid
as the sole source of carbon unless the medium also contains
a suitable H-acceptor, and nitrate is only one of several sub-
stances which can fulfil this function. Serratia marcescens
and Pr. vulgaris behave similarly [2, 4], but other organisms
are known which specifically use NO 7 and are unable to
grow anaerobically in its absence, even though the medium
contains NH3 . In anaerobic conditions, the chemosynthetic
autotroph, Thio. denitrificans, can obtain energy only by the
oxidation of sulphur compounds at the expense of reducing
NOI:

6KN03+5S + 2H20=K2S04+4KHS04+3N2+energy

A number of aerobic spore-forming bacilli related to
Bacillus subtilis can live anaerobically only in the presence
of NO 7, and they have been isolated from anaerobic enrich-
ment cultures in media containing a high concentration of
KNO3 (8-10 per cent) [5, 30].

The first step in the reduction of NO 7 involves its con-
version to NO 7 by an enzyme system which is adaptive in
nature, and is known as nitratase [29]. The nitratase of
Neurospora has a prosthetic group of adenine flavin di-
nucleotide and will use reduced TPN to reduce nitrate to
nitrite [zib]. Some strains oi Esch. coli are unable to reduce
NO 7 any further [24], but others reduce both NO 7 and
NO^ quantitatively to NH3 in the presence of a suitable
H-donor such as glucose [2, 36]. Organisms able to reduce

DENITRIFICATION 4I

NO 7 and which also possess hydrogenase can use molecular
hydrogen as a H-donor in these reductions [28]. Woods [36]
thus demonstrated that washed suspensions of CI. zvelchii
reduced NOJ, N07 and NH2OH to NH3 , the H2 uptake
being in accordance with the following equations:

NH^OH+H. = NH3+H.O
HNO,+3H2 = NH3+2H.,0
HNO3+4H, = NH3+3H2O

The Hg uptake in the third equation is that expected on
theoretical grounds if NH2OH and NOT are in fact inter-
mediates in the reduction of NO 7- During the early stages
of the reduction of NO^", a transient accumulation of NO^
was observed.

The recent investigations of Verhoeven [30] have done
much to confirm and extend the observations and hypotheses
of earlier workers in this field [13]. He found that the reduc-
tion of NO "3 by the aerobic spore-forming bacilli resulted in
the production of NO 7, Ng and sometimes NH3 ; NO 7
could be replaced as the H-acceptor by NOT or by NgO.
Strains producing large amounts of ammonia did not form
appreciable amounts of gaseous end-products, and the con-
verse was also true. A detailed study with one strain demon-
strated that the reduction process took place in two stages.
During the first stage NO 7 was converted to NOT and then
to NgO; whilst in the second, the gas evolved was mainly
N2 , indicating that NgO is the precursor of Ng . On two
occasions, Verhoeven detected NHgOH in denitrifying cul-
tures, and thus provided some evidence in support of
the contention of Blom that this compound is an inter-
mediate in the reduction of NO J. Working with Ps. stutzeri^
Allen and van Niel have come to the conclusion that, at
least in this organism, although NgO was reduced, it was
not a natural intermediate. They believe that nitramide
(NO2.NH2) is a possible intermediate since Ng was formed
from a preparation of this compound but not from hypo-
nitrous acid [i]. In the presence of a H-donor, cell-free ex-
tracts of Ps. stutzeri and B. subtilis reduce nitrate to Ng {Ps.

42

NITROGEN METABOLISM

stutzeri) or appreciable quantities of NgO {B. subtilis). Ex-
tracts of both organisms convert nitrite to nitric oxide and
nitric oxide to Ng [aifl]. Further advances in the elucidation
of the pathways of the biological reduction of nitrate await
the collection of more information concerning the chemical
properties of nitroxyl (HNO), hyponitrous acid (HgNgOg),
nitramide and other similar compounds of nitrogen, and
the development of unequivocal methods for their identifi-
cation and estimation. The scheme given below is based on
those proposed by Kluyver [cf. 30] and by Allen and van
Niel: it will be realized that there is no direct evidence that
nitroxyl or hyponitrous acid or nitramide is a natural
intermediate.

(R.NHo)

(NO2.NH2)
t
N2O

HNOa

HNO,

N,

>NH3

(HNO)

(H2N2O2)

-^N^O

Hypothetical Pathways of Nitrate Reduction in Micro-organisms

Routes 'a' and *6' may be operative in Ps. denitrificans,
and 'c' in the aerobic spore-forming bacilli [30]. Allen and
van Niel postulate that in Ps. stutzeri^ NO"^ enters into
organic combination and is reduced to an amino compound
(R.NH2) which then reacts with another substance, perhaps
NO"^ itself, to yield nitramide (route 'd').

The presence of oxygen tends to suppress the reduction
of no's, the degree of inhibition being determined both by
the partial pressure of Og and the organism concerned, e.g.
Esch. coli [29] is more sensitive than Ps. denitrificans [26].
It is interesting to note that restricted aeration of cultures
of the denitrifying bacilli resulted in the production of large
amounts of NH3 , even by those strains which in anaerobic

DENITRIFICATION 43

conditions produced large amounts of Ng and NgO. How-
ever, no such effect was obtained with Pseudomonas organ-
isms; they all produced Ng and no NHg . If cultures of
the Bacillus and Pseudomonas organisms were sufficiently
aerated there was no reduction of NO 7-

The ability to reduce nitrate has proved to be of value in
the classification and identification of micro-organisms, e.g.
in the yeasts, the genus Hansenula is differentiated from
Pichia on the basis that only the former can grow on NO 7
as a source of N. The gaseous products or nitrite formed by
the activities of nitrate-reducing organisms are responsible
for troublesome and unwelcomed consequences in certain
industries. Thus N07 and N07 are commonly used for
curing and preserving meat products and spoliation is often
due to denitrifying bacteria. Some workers believe that
denitrifying micro-organisms convert an appreciable amount
of fertilizers such as (NH4)2S04 and KNO3 into gaseous
products and thus significantly decrease the amount of
nitrogen available for plant growth [cf. 32]. After feeding on
oat hay, which frequently contains large amounts of NOs"*
sheep may show signs of methaemoglobinaemia (oat hay
poisoning) and this condition is due to the absorption of
NO 2" formed by micro-organisms in the rumen [17]. If
acidic conditions arise during the commercial production of
alcohol by the fermentation of sugar molasses, large amounts
of nitrogen peroxide are sometimes evolved. This is due to
the acidic decomposition of nitrites which were formed from
nitrates by micro-organisms during the processing of the
molasses [32].

REFERENCES

1. Allen, M. B. and van Niel, C. B. (1952), J. Bact., 64, 397 •

2. Aubel, E. (1938), C.R. Soc. Biol., Paris, 128, 45

3. Audus, L. J. (1946), Nature, 158, 419

4. Baas-Becking, L. G. M. and Parks, G. S. (1927), Physiol. Rev.

7,85

5. Beijerinck, M. W. and Minkman, D. C. J. (1910), Centr.

Bakt. Parasiteiik, (2. Abt.), 25, 30

6. Bomeke, H. (1939), Arch. MikrobioL, 10, 385

44 NITROGEN METABOLISM

7. Chick, H. (1906), Proc. Roy. Soc, 77B, 241

8. Frankland, P. F. and Frankland, G. (1890), Phil. Trans. Roy.

Soc, 181B, 107

9. Fred, E. B. and Davenport, A. (1921), Soil Sci., 11, 389

10. Hofman, T. and Lees, H. (1952), Biochem. J., 52, 140

11. Jensen, H. L. (1950), Nature, 165, 974

12. Kingma-Boltjes, T. Y. (1935), Arch. MikrohioL, 6, 79

13. Korsakova, M. P. (1927), Bull. Acad. Sci. U.S.S.R., Ser. 6,

1221

14. Lees, H. (195 1), Nature, 167, 355
/ 15. (1^52), Biochem. y., 52y 134

J ~ 16. and Quastel, J. H. (1946), Biochem. J., 40, 803, 815, 824

17. Lewis, D. (1951), Biochem. J., 48, 175; 49, 149

18. Meek, C. S. and Lipman, C. B. (1922), J. gen. Physiol., 5, 195

19. Meiklejohn, J. (1950), J. gen. Microbiol., 4, 185

20. Meyerhof, O. (1916), Pfliig. Arch. ges. Physiol., 164, 353; 165,

229; (1917), 166, 240
2.1a. Najjar, V. A. and Allen, M. B. (1954), J. hiol. Chem., 206, 209
2.1b. Nason, A. and Evans, H. J. (1953), ^ biol. Chem., 202, 655
21C. Nelson, D. H. (193 1), Zbl. Bakt. (2. Abt.), 83, 280
22. Quastel, J. H. and Scholefield, P. G. (195 1), Bact. Rev., 15, i
, 23. Scholefield, P. G. and Stevenson, J, W. (1952), Bio-
chem. J., 51, 278

24. and Stephenson, M. (1925), Biochem. J., 19, 660

^ 25. Stephenson, M. and Whetham, M. D. (1925), Biochem.

y-, 19, 304

26. Sachs, L. E. and Barker, H. A. (1949), J. Bact., 58, 11

27. Schloessing, T. and Muntz, A. (1877), C.R. Acad. Sci., Paris,

84, 301; 85, 1018

28. Stephenson, M. and Stickland, L. H. (1931), Biochem. y., 25,

205

29. Stickland, L. H. (1931), Biochem. y., 25, 1543

30. Verhoeven, W. (1952), Thesis: Delft, Aerobic sporeforming

nitrate reducing bacteria

31. Warington, R. (1891), J. chem. Soc, 59, 484

32. Wimmer, G. (1904), Z. Hyg. Infaktkr., 48, 135

33. Winogradsky, S. (1890), Ann. Inst. Past., 4, 213, 257, 760

34. (1891), Ann. hist. Past., 5, 32, 577

35. (1933), Ann. Inst. Past., 50, 350

36. Woods, D. D. (1938), Biochem. y., 32, 2000

CHAPTER IV

THE FIXATION OF NITROGEN

For several centuries it has been a matter of common
observation and agricultural practice that soil impoverished
by the growth of cereals can be revitalized by allowing the
land to lie fallow or by growing leguminous plants, yet not
until the end of the nineteenth century were adequate
explanations forthcoming as to why these procedures caused
such beneficial effects. They are in fact due to micro-
organisms which, either themselves or when in association
with leguminous plants, possess the ability to use the
atmosphere as a source of nitrogen. The conversion of mole-
cular nitrogen (N2) into nitrogenous compounds which can
be assimilated by the organisms concerned is termed nitrogen
fixation. The ability to fix Ng appears to be restricted to
micro-organisms and even amongst them it occurs in but
a few genera.

Priestley's claim that green plants absorbed N2 as well as
CO 2 and the suggestion of Sir Humphrey Davy that their
nitrogen might be derived from the atmosphere by the
agency of 'mushrooms and funguses' were the cause of
much controversy and stimulated several investigations
designed to test these statements by experiment [41].
Boussingault in 1838 was the first to show that, when they
were grown in sand, the nitrogen content of clover plants
increased yet that of wheat did not. Although in the years
that followed, the swellings or nodules invariably found in
the root systems of leguminous plants were frequently com-
mented upon, half a century was to elapse before the
classical experiments of Hellriegel and Wilfarth established
that they were the site of the agency which enabled legumin-
ous plants to fix atmospheric Ng . They found that nodules
were formed only in non-sterile environments and that in
contrast to cereals the growth of leguminous plants was
normal even in the absence of fixed nitrogen (i.e. nitrate,

45

46 NITROGEN METABOLISM

NHt, etc.) provided they were able to develop these struc-
tures. By this time nodule formation was known to be the
outcome of bacterial invasion of the root tissues [cf. 41, 35]
and the organisms living in them were described by Frank
under the name oi Bacterium radicicola [18]. Contrary to the
ideas of previous workers, Hellriegel and Wilfarth suggested
that the bacteria were not parasites but lived in symbiotic
association with the plant and endowed it with the ability
to grow at the expense of atmospheric Ng and thus be inde-
pendent of an exogenous source of fixed nitrogen. Pure
cultures of the nodular organisms, now placed in the genus
Rhizobium, were first isolated by Beijerinck, who also found
them free-living in the soil [3]. Like later workers, he was
unable to demonstrate that these organisms fixed Ng in the
absence of the host plant, and the mechanism of Na-fixation
by the symbiotic system still awaits elucidation. All the
strains of a given species of Rhizobium induce nodule forma-
tion in a restricted number of leguminous plants, termed a
cross-inoculation group, and it is on this basis that the
Rhizobium are classified into species, each species being
specific for one cross-inoculation group [41, i]. Although
nodules may be formed, they are not always effective, i.e.
capable of fixing nitrogen.

Isolation of free-living N ^-fixing organisms

During the latter half of the nineteenth century, Jodin
and Berthelot provided evidence that certain free-Hving
micro-organisms fixed atmospheric Ng , and pure cultures
of bacteria exhibiting this property were eventually isolated
by Winogradsky and Beijerinck. Each of these eminent
bacteriologists used the enrichment culture technique with
media which, apart from inorganic salts, contained only a
substance such as glucose or mannitol as a source of carbon
and energy: no nitrogenous compound was added. After
being inoculated with soil the cultures were incubated in an
atmosphere of air or nitrogen. Winogradsky thus isolated
the anaerobe Clostridium pasteurianum which fermented
glucose to acetic and butyric acids together with Hg and
CO 2 [49]. A few years later Beijerinck, using media

FIXATION OF NITROGEN 47

containing mannitol, isolated the two aerobes Azotohacter
chroococcutn and Azotohacter agilis (extremely motile) [4].
Apart from Az. indiciwi, Azotohacter spp. in general do not
fix Ng in an acidic environment, consequently their isolation
is facilitated by the use of a neutral or slightly alkaline
medium, e.g. one containing a buffer or CaCOg. Further-
more, the incorporation of a small amount of sodium
molybdate (5x10"® per cent) is frequently advantageous
since molybdenum appears to be of especial significance
in organisms which fix Ng .

Organic compounds, other than those which serve as
sources of carbon and energy, retard the growth of Azoto-
hacter but not Rhizohium. It is, however, unlikely that the
concentration of organic matter in the soil is ever great
enough to affect the fixation of Ng by Azotohacter in its
natural habitat. Of the thirty-five compounds tested only
aspartic acid, asparagine, glutamic acid, urea and adenine
replaced molecular Ng as a source of N for Azotohacter [20].
On the other hand, the growth of Rhizohium spp. was sup-
ported by any one of thirty-two organic nitrogen compounds
and was luxuriant in rich organic media [30]. The optimal
growth of fast-growing, but not slow-growing, strains of
Wiizohium is dependent on an exogenous supply of a sub-
stance originally named coenzyme R and later identified as
biotin [cf. 40].

Detection of N ^-fixation

Although from time to time the power to fix N 2 has been
attributed to many other organisms, the majority of these
claims must now be discounted on the grounds of faulty
experimentation. It is very difficult, if not impossible, to
eliminate all nitrogenous compounds from the materials
used to make media, consequently growth in what is believed
to be, apart from molecular Ng , a N-free medium is not a
sufficient criterion for stating that an organism can fix
nitrogen and, indeed, there is often no mention of control
cultures incubated in the absence of molecular Ng . It is
therefore relevant to consider the techniques used in the
detection and quantitative study of Ng-fixation. Apart from

48 NITROGEN METABOLISM

growth on a N-free media, these techniques involve gaso-
metric analysis, nitrogen estimations by Kjeldahl proce-
dures, manometry or the use of the nitrogen isotope N^^.

Gasometric analysis is used to detect whether there has
been a decrease in the amount of gaseous Ng in a closed
system, whilst increases in fixed nitrogen can be determined
by the use of a Kjeldahl technique. Although often used
successfully [22], gasometric analysis is tedious and the
elimination of experimental errors is not easy. The Kjeldahl
method, which at first sight appears to be an ideal and
technically simple procedure, has yielded many erroneous
results, the reasons for which have been cogently assessed
by Wilson [41]. The samples taken for analysis do not have
a homogenous composition, and whilst a particular Kjeldahl
procedure will estimate with precision the nitrogen content
of one or more related compounds, it does not necessarily
follow that the nitrogen in other compounds is estimated
with a comparable degree of accuracy; e.g. the mere addition
of water to dry seeds appeared to increase their nitrogen
content as measured by the Kjeldahl procedure being used.
None the less, provided such sources of error are borne in
mind, the Kjeldahl technique has been, and is, of great
value.

A more convenient, though indirect, method for study-
ing Ng-fixation by free-living organisms is based on the use
of the Warburg manometer, organisms being grown in the
flask of the apparatus and the gas changes consequent on
growth being followed in the usual manner. An obvious com-
plication with aerobic organisms is that superimposed on
any uptake of Ng , there is a concomitant uptake of Og due
to respiration. Indeed, Azotobacter and Rhizohium possess the
highest respiration rates known, the Qq^ on glucose being
of the order 1,000 to 2,000. Where large O2 uptakes are
expected, the usual Brodie manometric fluid is often re-
placed by one of greater density [e.g. Hg] in order that the
manometer readings will be of convenient dimensions. If
the rate of Ng-fixation is the factor limiting growth, then
fixation by aerobic organisms can be followed in terms of an
increase in the respiration rate, since the latter is directly

FIXATION OF NITROGEN 49

proportional to the mass of cytoplasm present in the
system. With Azotohacter, the Ng uptake is insignificant
compared with the O2 uptake, and is therefore neglected in
calculating the results. Direct proof that such assumptions
are justified was provided by Burk and Meyerhof, the
originators of this technique [29, 7]. In their experimental
conditions, irrespective of whether growth was followed by
nephelometry, dry-weight measurements or total cell counts,
the grov^h curves were all reasonably superimposable on
the graph relating respiration rate with age of culture.
Manometry has since been applied to the study of Na-fixa-
tion by anaerobic bacteria, such as Clostridia, Desulpho-
vibrio and photosynthetic organisms [34].

Unequivocal evidence as to whether a system can fix
N2 is provided by the use of N2 enriched with the non-
radioactive isotope N^^, a technique first introduced by
Burris [9] and which later proved to be of value in the
elucidation of the route of N2-fixation. The requisite control
experiments showed that non-enzymic exchange reactions
between molecular N2^^ and compounds containing nitrogen
were insignificant, and that in vivo there was no preferential
uptake of one isotope over another, i.e. the ratio of the
isotopes one to another in the gas-phase did not change
throughout the course of the experiment. To detect Ng-
fixation, the experimental material is placed in a closed
system through which N2 enriched with N^^ is circulated by
a Urey pump. The nitrogen-containing compounds of the
experimental material may be fractionated prior to being
converted into N2 for analysis in the mass spectrograph.
The technique is very sensitive and an increase of o-oi atom
per cent N^^ above the normal distribution of 0-36 is
regarded as being significant, and in certain conditions it is
possible to detect the fixation of o-ooi //g. N [9, 10].

Organisms fixing N2

In addition to CI. pasteurianuni and Azotobacter spp., there
is now adequate evidence, in many cases based on experi-
ments with N^^, that Ng-fixation is a property of several
other species of Clostridia [32], various photosynthetic

50 NITROGEN METABOLISM

bacteria (species of Rhodospirillum, RhodopseudomonaSy
Rhodomicrobium, Chromatium and Chlorohacterium) [19, 26
27, 28], Desulphovibrio [34], blue-green algae of the family
Nostocaceae [15, 17, 43], and Calothrix [43]. Claims for
fixation by yeasts and other fungi are as yet unsubstantiated.
The successful demonstration that nodules detached from
roots fix N2 was dependent on the use of nodules from
leguminous plants grown in the field rather than the green-
house, performing the experiments immediately after
detachment from the roots and subjecting only the soluble
nitrogen compounds of the nodules to isotopic analysis [2].
The Leguminoseae is not the only family in which nodules
are formed in consequence of microbial invasion, and the
root nodules of the Alder {Alniis) and Coriaria japonica may
also be concerned with the fixation of Ng.

Factors affecting Nz-fixation

There are no substantiated experiments in which Ng-
fixation has been divorced from growth of the experimental
material [24], consequently care is required in assessing
whether the factor being studied directly affects the fixation
mechanism: the observed effects may be no more than the
result of interference with processes, such as the production
of energy, which are essential to metabolism in general.
For example, the influence of the partial pressure of oxygen
(pOa) on fixation by Azotohacter and by clover plants
appears to be entirely explicable in terms of its effects
on respiration, and in consequence, the availability of
energy [7, 41]. Similarly, although molybdenum, iron,
calcium and strontium have all been implicated in the
fixation mechanism, it is difficult to decide whether this is
their primary function. These difficulties cannot be circum-
vented by performing experiments in the presence of fixed
nitrogen, since the latter induces a quicker rate of growth,
and in all probability growth is then limited by different
factors from those operative when the organisms are fixing
molecular Ng . Whilst molybdenum undoubtedly influences
the growth of Azotohacter^ Nostoc, CI. pasteurianum and
leguminous plants, there is no direct evidence that it is

FIXATION OF NITROGEN 5I

specifically concerned with the fixation mechanism (for
discussion and references concerning trace element nutri-
tion, see 8, 44, 21, 6).

Burk was the first to demonstrate that the rate of Ng-
fixation by Azotobacter was a function of the partial pressure
of Ng (pN2)» the relationship between the two conforming
to that expressed by the Michaelis-Menten equation for an
enzymic reaction. The pNg at which fixation occurred at
half the maximal rate (i.e. K^) was 0-21 atm., an unex-
pectedly high value for a gaseous substrate [7]. In order to
prepare gas mixtures containing different pNg , Burk had
used Hg as a diluent gas since at that time Hg was thought
to be inert in biological systems. Later experiments, first
with red clover plants [41, 47] and subsequently with
Azotobacter [50], revealed that Hg inhibited Ng-fixation and
increasing the pHg caused a progressive decrease in the
rate of growth: Hg had no effect on growth in the presence
of fixed nitrogen, e.g. (NH4)2HP04 . Helium and argon
exhibited no such inhibitory action, and in the absence of
Hg , fixation by Az. vinelandii was maximal at a pNg of
0-15 atm. and half maximal at 0-02 atm. (Fig. 4.1). Hence,
Wilson suggested that in natural conditions the pNg is not
a factor limiting fixation. By comparing the rate of grov^1:h
in air with that in gas phases containing, in addition to Og ,
either 0-15 atm. Ng or 0-3 atm. Ng , together with different
amounts of Hg , Wyss and Wilson deduced that Hg inhibited
fixation in a competitive manner. Unlike Azotobacter, Hg is
a normal product in fermentations by CI. pasteurianum, and
although the pHg did not influence the rate of Ng-fixation
by this organism it did affect the amount of Ng fixed and
the efficiency of the fixation process (i.e. mg. N fixed/g.
glucose fermented) [32]. Wilson has noted that all the free-
living organisms shown to fix Ng also possess the enzyme
hydrogenase [cf. 42]. He suggests that the latter may be
involved in the fixation mechanism, since the hydrogenase
activity of Azotobacter is greatly reduced, even in the
presence of Hg , w^hen growth is no longer dependent on
the fixation of molecular Ng [25]. Hydrogenase has not been
detected in Rhizobium either free-living or symbiotic, or in

52 NITROGEN METABOLISM

nodular tissues [45]. Nitrogen-fixation by Azotobacter,
Nostoc and leguminous plants is inhibited in an apparently
non-competitive manner by carbon monoxide in concentra-
tions which cause little, if any, inhibition of respiration [48,
16]. Carbon monoxide is an isostere of Ng , and it may
therefore replace Ng on the surface of the enzyme concerned

100-

^

r^

X.

'0

>^-^

-0

?80-
<

/

y

/^

2

J

^/

/^

Ui

1-
<
^ 60-

0

/

^

J

^

^ /

1

|40-

a.

I

FIXATION

0
1

/

/ 0-7
/ 1

0-6

1

o-s

0-4 pH2 or Inert gaJ

_!

0-

/

o.f.

0)3

0.4 P^zC'" at'"-)

_J

FIG. 4.1. — Nitrogen fixation by Azotobacter as a function of the
partial pressure of nitrogen (pNo) in the presence of different
partial pressures of hydrogen (6), helium ( X ), or argon (A),
or a partial vacuum (D)"- partial pressure of oxygen in all
experiments, 0*2 atm. [50]

with the primary fixation reaction. Alternatively the fixation
mechanism may involve a metal ion, perhaps combined
in the prosthetic group of an enzyme, whose activity is lost
on combination with CO. In this connection it may be of
significance that the hydrogenase of Az. vinelandii is also
inhibited by CO [44].

Role of ammonia in N^-jixatioyi

The idea has arisen that there is a key substance in

FIXATION OF NITROGEN 53

nitrogen-fixation, one which can be regarded both as the
product of the fixation mechanism and as the substrate for
the reactions by which inorganic nitrogen is incorporated
into organic compounds. During the last decade, the eluci-
dation of the nature of this substance has occupied the
attention of two groups of workers, one associated with
Virtanen in Finland [36] and the other with Burris and
Wilson in the U.S.A. By using the isotope N^^, the American
workers have obtained convincing evidence of the key role
of NH3 in the fixation of N2 , a concept first advanced by
Winogradsky [49]. In the isotope experiments, the period
of exposure to the substance enriched with N^^ was not
long enough for equilibrium to be established, and prior to
isotopic analysis, the nitrogenous components of the experi-
mental system were separated into fractions to facilitate
location of the compounds with the highest concentration
of N^^. For example, after acid hydrolysis, various amino-
acids were isolated by classical precipitation procedures, or
more recently, by the use of columns of ion exchange resins
or starch [51, 43]. It was argued that if the assimilation of
nitrogen involved a number of intermediates, then, before
equilibrium was established, the concentration of the iso-
tope would be the greater the nearer the intermediate to the
substance initially enriched with N^^. Moreover, if a sub-
stance on the fixation pathway was supplied instead of, or
in addition to, molecular Ng , it should be utilized not only
as a source of nitrogen, but also in preference to its pre-
cursors (cf. simultaneous adaptation, p. 13) [11, 44].

The resultant distribution of the isotope amongst the
constituents of Az. vinelandii was the same irrespective of
whether nitrogen was derived from Ng^^ or N^^Ht, and of
the cells' amino-acids, glutamic acid followed by aspartic
acid, contained the highest concentration of N^^ [12]. The
establishment of such a distribution, even when the period
of contact with N^^Ht was short (3 min.), indicated that the
enzymes responsible for the assimilation of NHg already
existed in the bacteria. Moreover, the fact that NH3 gave
rise to the same distribution of N^^ as N2 ^ implied that it
was either itself on the pathway of Ng-fixation or at least

54 NITROGEN METABOLISM

closely related to one of the natural intermediates. The high
concentration of N^^ in glutamic acid was indicative of its
importance in the pathway of Na-fixation. Such a conclusion
was in keeping with the results of previous work with other
organisms in which it had been established that glutamic
acid occupies a key position in amino-acid metabolism and
could be synthesized from NHg and a-ketoglutarate by the
glutamic acid dehydrogenase system (cf. Chap. II).

Winogradsky was the first to note, during his experiments
with CI. pasteurianum, that ammonium salts inhibited N2-
fixation. Azotobacter responds in a similar fashion, both to
ammonium salts and to compounds (urea, asparagine) which
it can convert to NH3 [46]. Since nitrate and nitrite were
only inhibitory after a lag period, it was inferred that they
were not utilized by Azotobacter until the appropriate
enzymes had been formed by adaptation, and in fact there
was no lag period with cells which had been grown in media
containing NO7 and NO^ • Organic nitrogen compounds,
e.g. aspartic and glutamic acids, were only moderately
inhibitory, perhaps not unexpectedly since they are probably
more concerned with intermediary metabolism rather than
with the initial steps of the fixation mechanism.

Further isotope experiments provided additional evidence
that the fixation of N 2 by CI. pasteurianum, Chromatium and
Nostoc muscorum is accomplished by essentially the same
route as in Az. vinelandii [43]. In each of these organisms
after exposure to Ng-^^ or N^^H4 , the dicarboxylic amino-
acids, and in particular glutamic, contained the highest
concentration of N^^. Furthermore, the results of the ex-
periments with CI. pasteurianum. [51] proved to be in some
respects comparable with those of Virtanen with nodulated
leguminous plants. Under certain conditions, the fixation
of N 2 by CI. pasteurianum and by the symbiotic system was
accompanied by the excretion of nitrogenous compounds
into the environment; the anaerobe excreted mainly NH3
whilst the plant excreted aspartic acid, ^-alanine and a small
amount of an oxime, identified as oximinosuccinic acid.
These substances are regarded as being products, not of
catabolism, but of the processes directly concerned with

FIXATION OF NITROGEN 55

Ng-fixation. After exposing cultures of CI. pasteurianum in
the log phase of growth to Ng^^, the concentration of N^^
in the NH3 isolated from the medium was greater than that
of the amide nitrogen of the cell protein which was in turn
greater than the average level of the isotope in the pro-
teins as a whole. This indicated that the excreted NH3
was a product of the fixation processes and did not arise
by deamination of amino-acids [51]. The excretion of NH3
only occurs when the organisms are grown in certain media,
and it is apparently an expression of a deficiency, probably
of suitable organic acceptors, since supplementing the
medium with biotin, ^-aminobenzoic acid and a-keto-
glutarate completely stopped the excretion of NH3 and yet
had little effect on the rate of N2-flxation [43].

Role of hydroxylamine in N2-fixation

Because leguminous plants apparently excreted only
aspartic acid and substances related to it [38, 5], Virtanen
originally concluded, especially in view of the presence of
the oximino-compound, that hydroxylamine (NHgOH) was
the key product of the fixation mechanism. He proposed
that NH2OH condensed with oxaloacetate, a substance
known to be present in nodulated roots, thus forming
oximinosuccinic acid which was then reduced to aspartic
acid. Many other workers were unable to repeat these experi-
ments, and following a successful visit to Virtanen's labora-
tory, Wilson concluded that excretion was observed only
when the rate of photosynthesis was not sufficient to supply
enough materials for the utilization of all the products of
the fixation mechanism [41]. The later discovery [39] that
glutamic acid was present amongst the excreted substances
implied that many of the results of the leguminous plant
experiments could also be interpreted in favour of the
importance of NH3 in the fixation mechanism. Whilst it
seems most probable that nitrogen enters into organic com-
bination in the form of NH3 and not NHgOH, the possi-
bility that NHgOH is a precursor of the NH3 , and perhaps,
in certain circumstances, reacts directly with an organic
acceptor, has not yet been excluded [cf. 37].
5

56 NITROGEN METABOLISM

A series of well-controlled experiments has failed to find
any evidence for the participation of NHgOH in the fixation
of N 2 by Azotohacter and CI. pasteurianum [31, 33]. Hydrox-
ylamine in concentrations greater than about 2 /<g./ml. was
toxic, and growth, when it occurred, could be accounted for
in terms of NH3 produced by the spontaneous decomposi-
tion of NHgOH. Even with Ni^HgOH the results were diffi-
cult to interpret because the amount of nitrogen involved
was too small to permit significant determinations. The
oximes of a-ketoglutarate, oxaloacetate and pyruvate were
slowly utilized by CI. pasteurianum but not by Azoto-
hacter [34].

Pathways of N^-fixation

There has been little support in recent years for the
fixation of nitrogen being explained in terms of an oxidative
pathway involving compounds such as nitrous oxide and
hyponitrous acid. There is now a considerable amount of
evidence that it is probably a process involving a number of
reductive mechanisms which terminate in the formation of
NH3 . Whilst the steps between molecular Ng and NH3 are
completely unknown some hypothetical pathways are shown
in Fig. 4.2 (details of other schemes will be found in
reference 44). Although the fixation of Ng by cell-free
systems has yet to be confirmed, past failures [24] may be
attributed, at least in part, to the harsh procedures employed
in the preparation of the extracts.

In spite of their diverse nature, bacteria, blue-green algae
and leguminous plants all apparently fix nitrogen by
mechanisms which, if not identical, have at least many
features in common [43]. They all respond in a similar
manner to changes in the partial pressure of Ng, Hg and
CO and their K^ values for Ng are of the same order. In
view of the repeated failures to demonstrate Ng-fixation by
free-living cultures of Rhizobium, interest with regard to the
symbiotic system is centred around the question why
association with the leguminous plants should endow one
or other of the symbionts with this ability. A noticeable
feature of the association is the presence of a red haemo-

FIXATION OF NITROGEN

57

globin-like pigment ('leghaemoglobin') in the effective
nodules [44]. The pigment is only formed after the nodules
have been established and concurrent with the change to a
green colour, their ability to fix N2 declines. The pOg in the
nodules is relatively low and the pigment may serve to store
and transport Og to the Rhizobia, which, it will be remem-

•NEN

+ H

nitrous oxide

+ H2P

nitrogen

+ HP
/
(NOH), HO. N.N. OH
hyponitrous acid /H H

nitrogen

+ H

NH^OH_
hydroxylomine

+ COCOOH

I

CH^COOH
oxaloacetic
acid

hooc.ch2c(noh) cooh

oximinosuccinic acid

H00C.CH2CH(NH2.)C00H
ospgrtic acid

(CH2)2C00H +

COCOOH
oC-ketoglutaric
ocid

(CHp)2COOH

ch(nh2)cooh

glutamic acid

FIG. 4.2. — ^Hypothetical pathways for the fixation of
molecular nitrogen

bered, possess a high rate of respiration. Some workers have
suggested that the haemoglobin participates directly in the
ifixation mechanism. For example, NHgOH is decomposed
by haemoglobin (Hb) in vitro, thus [14]:

NH20H+2Hb+++H20 -> 2MetHb++++NH3+20H-
2NH20H+2MetHb+++ -> 2Hb+++2H20+N2+2H+
and it is possible that the fixation of nitrogen is accom-
plished by the reverse of these reactions. However, claims
for the natural occurrence of methaemoglobin in the nodules

58 NITROGEN METABOLISM

have been seriously challenged [23]. The addition of haemo-
globin increases the respiration of i^feo^mw, but this appears
to be an indirect effect [13] and not connected with the
transport of O2 .

It will be evident that there are still many aspects of
nitrogen fixation to be explored, and apart from their bio-
chemical interest, their economic importance should not be
underestimated. The growth of plants, the leaching effects of
rain-water and the activities of denitrifying organisms all
tend to remove from the soil the nitrogenous compounds
which are essential to the continued existence of most forms
of plant life. This loss is in part restored naturally by the
decomposition of plants and animals, and artificially by the
application of inorganic or organic fertilizers. Fixed nitrogen
compounds produced commercially probably account for no
more than 15% of the nitrogen returned annually to the soil.
By far the greatest proportion is due to Na-fixation by bio-
logical agents, and it has been estimated that symbiotic
systems and free-living organisms are responsible respectively
for returning to the soil 5-46x106 and 4-37x10^ tons of
nitrogen per year, yet, even allowing for this, there appears
to be an annual overall loss in soil nitrogen [42].

REFERENCES

1. Allen, E. K. and Allen, O. N. (1950), Bad. Rev., 14, 273

2. Aprison, M. H. and Burris, R. H. (1952), Science, 115, 264

3. Beijerinck, M. (1888), Bot. Zbl, 39, 356

4. (1901), Zbl. Bakt., II, 7, 561

5. Billen, D. and Lichstein, H. C. (1949), J. Bad., 57, 267

6. Bortels, H. (1940), Arch. Mikrobiol., 11, 155

7. Burk, D. (1934), Ergeb. Enzymforsch., 3, 23

8. and Burris, R. H. (1941), Ann. Rev. Biochem., 10, 587

9. Burris, R. H. (1942), J. biol. Chem., 143, 509

10. Eppling, F. J., Wahlin, H. B. and Wilson, P. W. (1943),

J. biol Chem., 148, 349

II. and Wilson, P. W. (1946), J. Bad., 52, 505

12. (1946), J. biol. Chem., 165, 595

13. • (1952), Biochem. J., 51, 90

14. Colter, J. S. and Quastel, J. H. (1950), Arch. Biochem., 27, 368

15. De, P. K. (1939), Proc. Ro\. Soc. Lond., 127B, 121

16. Ebersole, E. R., Guttentag,"C. and Wilson, P. W. (1944), Arch.

Biochem., 3, 399

FIXATION OF NITROGEN 59

17. Fogg, G. E. (1947), Endeavour, 6, 172

18. Frank, B. (1879), Bot. Ztg., 37, 377, 393

19. Gest, H., Kamen, M. D. and Bregoff, H. M. (1950), jf. biol.

C/iem., 182, 153

20. Horner, C. K. and Allison, F. E. (1944),^. Bad., 47, i

21. Burk, D., Allison, F. E. and Sherman, M. S. (1942),

y. agric. Res., 65, 173

22. Hurwitz, C. and Wilson, P. W. (1940), Ind. Eng. Chem. Anal.,

ed. 12, 31

23. Keilin, D. and Smith, J. D. (i947), Nature, 159, 692

24. Lee, S. B., Burris, R. H. and Wilson, P. W. (1942), Proc. Soc.

exp. Biol. N.Y., 50, 96

25. and Wilson, P. W. (1943), J. biol. Chem., 151, 377

26. Lindstrom, E. S., Burris, R. H. and Wilson, P. W. (1949), J.

Bact., 58, 313

27. Lewis, S. M. and Pinsky, M. J. (1951), J. BacL, 61, 481

28. Tove, S. R. and Wilson, P. W. (1950), Science, 112, 197

29. Meyerhof, O. and Burk, D. (1928), Z.phys. Chem., ly^P^, J 17

30. Nielsen, N. (1940), C.R. Lab. Carlsberg, 23, 115

31. Novak, R. and Wilson, P. W. (1948), J. Bad., 55, 517

32. Rosenblum, E. D. and Wilson, P. W. (1949), J. Bad., 57,413;

(1950), 59, 83; (1951), 61, 475

33. Segal, W. and Wilson, P. W. (1949), J- Bad., 57, 55

34. Sisler, F. D. and Zobell, C. E. (195 1), Science, 113, 511

35. Thornton, H. G. and Nicol, H. (1936), Nature, 137, 494

36. Virtanen, A. L (1947), Biol. Rev., 22, 239

37. and Hakala, M. (1949), Acta chem. scand., 3, 1044

38. and Laine, T. (1939), Biochem. J., 33, 412

39- Linkola, H., Hakala, M. and Rautanen, N. (1946),

Suomen Kemistilehti, 19B, 83

40. Wilson, J. B. and Wilson, P. W. (1942), J. Bact., 43, 329

41. Wilson, P. W. (1940), The Biochemistry of Symbiotic Nitrogen

Fixation, Wisconsin U.P., Madison, U.S.A.

42. (195 1 ), in Bacterial Physiology, Chap. 14

43- (1952), Advances in Enzymology, 13, 345

44- and Burris, R. H. (1947), Bact. Rev., ii, 41

45- Burris, R. H. and Coffee, W. B. (1943), J. biol. Chem.,

147. 475

46. Hull, J. F. and Burris, R. H. (1943), Proc. Nat. Acad.

Sci., Wash., 29, 289
47- Lee, S. B. and Wyss, O. (1941),^ biol. Chem., 139, 91

48. and Lind, C. J. (1943), jf. Bact., 45, 219

49. Winogradsky, S. (1893), C.R. Acad. Sci., Paris, 116, 1385;

(1894), 118, 353

50. Wyss, O., Lind, C. J., Wilson, J. B. and Wilson, P. W. (1941),

Biochem. J., 35, 845

51. Zelitch, L, Rosenblum, E. D., Burris, R. H. and Wilson, P. W.

(1951), J- biol. Chem., 191, 295; (1951), J- Bad., 62, 747

CHAPTER V
SYNTHESIS OF AMINO-ACIDS

From the vast amount of information now available it is
clear that glutamic acid and aspartic acid occupy a key
position in amino-acid metabolism, and, of the two, the
former is the more important. Glutamic acid can be synthe-
sized from NH3 and a-ketoglutaric acid by the glutamic
dehydrogenase system (p. 12) and aspartic acid from NH3
and fumaric acid by aspartase (p. 23). The direct addition
of an inorganic compound of nitrogen to the appropriate
carbon skeleton does not appear to be a general route for
the synthesis of amino-acids, and the importance of the
dicarboxylic amino-acids is in part due to the fact that they
contain nitrogen in a form which can be transferred to suit-
able acceptors and thus utilized for the synthesis of other
nitrogenous compounds, e.g. the conversion of citrulline to
arginine (p. 70) and the synthesis of amino-acids by trans-
amination. Moreover, it will become clear from the following
paragraphs that several amino-acids may be derived from
other preformed amino-acids — for example, proline and
ornithine can be synthesized from glutamic acid (pp. 69, 70).
Many organisms use an inorganic form of nitrogen, such as
molecular N2 , NH3 and NOJ, as a source of this element,
and it is now generally held that the first steps in the
utilization of molecular Ng and nitrate for this purpose
involves their conversion to NH3 (see Chaps. IV and III).
It is therefore worthy of note that the glutamic dehydro-
genase system and aspartase are mechanisms which enable
inorganic nitrogen in the form of NH3 to be incorporated
into an organic molecule.

Transamination and amino-acid synthesis

A transaminase catalyses the reversible transfer of the
amino group of one amino-acid to the a-keto acid corre-
sponding to another amino-acid:

60

SYNTHESIS OF AMINO-ACIDS 6l

RiCH(NH,)COOH+R2COCOOH ^

RiCOCOOH+R2CH(NH2)COOH (i)

Reactions of this type were first discovered in animal tissues
by Braunstein and Kritzmann, who concluded that the
amino groups of several amino-acids could be transferred
in such a manner provided one of the participants in the
system was a dicarboxylic acid, i.e. aspartic, glutamic,
oxaloacetic, or a-ketoglutaric acid [4]. Cell-free preparations
of what were believed to be two distinct transaminases were
obtained, one catalysed reactions ii and iii, and the other
reaction iv:

glutamate+ pyruvate ?^ a-ketoglutarate+ alanine (ii)

glutamate+oxaloacetate ^^ a-ketogIutarate+ aspartate (iii)

aspartate + pyruvate ?=i oxaloacetate+ alanine (iv)

Later workers isolated two enzyme systems, specific for re-
actions ii and iii respectively, and Kritzmann 's second trans-
aminase was shown to be an artifact and due to a mixture
of these two enzymes with catalytic amounts of glutamate
functioning as a carrier between the two systems [23].

Similar transaminase systems were later found in bacteria,
yeasts [39^] and Neurospora [17^, Z>]. Washed cell suspensions
of various bacteria (staphylococci, streptococci, pneumo-
cocci, enterobacteria, Az. agilis, and Ps. pyocyanea) cata-
lysed the transfer of the amino group of glutamic acid to
oxaloacetic acid (reaction iii), and cell-free preparations of
Strep, faecalis accomplished both reactions ii and iii, the
equilibrium being in favour of the synthesis of alanine and
aspartic acid respectively [34, 35]. Like the animal trans-
aminases, the bacterial enzymes possess a prosthetic group
of pyridoxal phosphate. The glutamic-aspartic trans-
aminase of Strep, faecalis had been partially resolved and
activity was restored by the addition of synthetic pyridoxal
phosphate or natural codecarboxylase. The advent of paper
chromatography has facilitated the detection and identifica-
tion of small quantities of amino-acids and has been used
to demonstrate that several amino-acids can transfer their
amino groups to a-ketoglutarate. These experiments have
been performed with Esch. coli, Ps.fluorescens, B. suhtilis [14]

62 NITROGEN METABOLISM

and Lb. arahinosus [36a] and also with animal tissues [26].
Unequivocal proof that such results are due to transamina-
tion awaits the isolation of the appropriate enzymes, and,
indeed, a glutamic-tyrosine transaminase and a glutamic-
phenylalanine transaminase have been isolated from Esch.
coli and shown to contain prosthetic groups of pyridoxal
phosphate [14]. All known transaminases are specific for the
L-isomers of the amino-acids.

Our conceptions of the mechanism operative in biological
transamination are based on analogy with in mtro systems.
Transamination is a true transfer process and there is no
evidence at all that the amino group becomes free as NH3 at
any stage in the reaction. Herbst has proposed that a type
of Schiif's base is formed by condensation between the
amino and keto groups of the two substrates and that this
is followed by a molecular rearrangement involving the
alpha hydrogen atom of the amino donor, after which the
base is ruptured by hydrolysis [25]. Incubation of alanine
containing deuterium in the alpha position with a-keto-
glutarate and a mammalian glutamic-alanine transaminase
resulted in the rapid appearance of deuterium in the water
of the experimental system and is evidence in favour of
such a mechanism:
Ri R2 Ri R2

I I I I ■
CH(NH,)+CO ^HC— N = C ^

I " I II
COOH COOH COOH COOH

Ri R2 Ri R2

I I I 1

C = N— CH ^ CO + CH(NH,)

I I I I "

COOH COOH COOH COOH

It is to be remembered that pyridoxal phosphate itself con-
tains a carbonyl group and Snell, on the basis of non-
enzymic chemical experiments, has made the following
proposal [42]:

amino-acid A+pyridoxal phosphate ^

keto acid A+pyridoxamine phosphate
pyridoxamine phosphate + keto acid B ^^

amino-acid B+pyridoxal phosphate

t

SYNTHESIS OF AMINO-ACIDS 63

Synthetic pyridoxamine phosphate activated some prepara-
tions of partially resolved bacterial transaminases [48], and
though first reported not to restore activity to a resolved
preparation of the pig heart glutamic-aspartic transaminase,
later workers showed that it was as active as pyridoxal phos-
phate provided it was incubated with the apoenzyme for
30-60 minutes before adding the substrates [366]. Gunsalus
and Tonzetich have recently demonstrated that prepara-
tions oiEsch. coli catalysed transamination reactions between
pyridoxal and glutamic acid and between pyridoxamine and
a-ketoglutarate. Hence, while there is some reason to believe
that the prosthetic group of the transaminases can function
a£ a carrier of amino groups, direct proof that it does so has
still to be obtained.

Owing to the lack of essential data, it is not yet possible
to assess whether transamination is of key importance in
amino-acid synthesis. The available information, admittedly
extremely incomplete, indicates that the transaminases are
widely distributed in micro-organisms, at least in those non-
exacting to amino-acids. Whether or not there is a complete
series of enzymes capable of reacting with the keto acids
corresponding to all the natural amino-acids is still not
known. If transamination is involved in the synthesis of an
amino-acid, then it follows that the organism must be able
to synthesize the appropriate keto acid. Except for pyruvate,
oxaloacetate and a-ketoglutarate, virtually nothing is known
about the synthesis of these compounds, and although the
two former acids are known to be produced in the inter-
mediary metabolism of many organisms, conclusive evidence
of the ability to synthesize a-ketoglutarate is only available
for Ps. fluorescens, Az. agilis and Sac. cerevisiae. Although in
recent years it has been thought that glutamic acid was the
only amino-acid able to transfer an amino group to a wide
variety of keto acids, there is now some evidence that
aspartic acid can transaminate with acids other than a-keto-
glutarate, e.g. there is an aromatic amino-acid — aspartic
acid transaminase in Esch. coli [39^]. Furthermore, trans-
amination reactions are now known in which dicarboxylic
acids do not participate, e.g. Brucella abortus appears to

64 NITROGEN METABOLISM

possess a leucine-alanine transaminase [ic]. Transamination
can therefore be visualized as being of some significance in
amino-acid synthesis provided the organism can either syn-
thesize one or more amino-acids from ammonia by a direct
route (e.g. the glutamic acid dehydrogenase system or
aspartase), or obtain suitable amino group donor amino-
acids from the environment.

Metabolite analogues and the elucidation of metabolic pathways
Many enzyme systems are inhibited in a competitive
manner by substances similar in chemical structure to their
normal substrates: such substances are known as metabo-
lite analogues and have been used, for example, to study
the synthesis of tryptophan by Salmonella typhosa [15].
Although a reaction inhibited in this fashion may be essential
for grow^th, nevertheless, growth may be possible provided
the medium is fortified in some way, i.e. by the addition of
substances antagonizing the effects of the inhibitor. Ideally,
the substrates of the inhibited reaction act as competitive
antagonists, whereas the products act in a non-competitive
manner. The grow^th of freshly isolated strains of Salm.
typhosa is dependent on tryptophan or indole, but non-
exacting strains frequently arise. The inhibitory effects of
/?-indoleacrylic acid on the growth of a non-exacting strain
were completely overcome by the addition of tryptophan.
Indole accumulated in the media of non-exacting strains
growing in the presence of limited amounts of tryptophan
and sub-lethal amounts of indoleacrylic acid, and was pre-
sumed to be the substrate of the reaction blocked by the
inhibitor. Serine was a powerful antagonist and high con-
centrations of this amino-acid also decreased the accumula-
tion of indole. Fildes therefore concluded that tryptophan
was synthesized by the condensation of serine with indole
and that indoleacrylic acid inhibited this reaction. The work
of Snell and Schweigert had already indicated that an-
thranilic acid might be an intermediate in the synthesis of
tryptophan by Lb. casei and Lb. arabinosus, and it was later
found that irrespective of their tryptophan requirements,
strains of Salm. typhosa secreted anthranilic acid into the

SYNTHESIS OF AMINO-ACIDS 65

culture medium [40]. Since the amount of anthranilic acid
produced by an exacting strain was greater than the amount
of indole utilized, it was unlikely that the former was derived
from the latter. The inhibitory effect of 4- or 5-methylan-
thranilic acid on the growth of the non-exacting strain was
reversed by anthranilic acid, indole or tryptophan [16].
These results are summarized in the following scheme,
vertical arrows denoting the site of action of inhibitors:

4-methyl- indoleacrylate, methyl-

anthranilate 4-methylindole tryptophan

anthranilic ] >■ indole"! |

acid Y j *■ tryptophan '

serineJ y

'*" protein

Another example of antagonism in amino-acid metabolism
comes from studies of the nutrition of Bacillus anthracis and
serves to emphasize that growth may be dependent not only
on the presence of certain amino-acids but also on their
relative concentrations [19]. Although B. anthracis would
not grow on a synthetic and complete amino-acid medium
from which valine, leucine or isoleucine had been removed,
growth did occur in the absence of all three of these amino-
acids. When valine was present, no growth was possible
unless a suitable amount of leucine had also been added,
whilst growth in the presence of isoleucine was conditional
on the addition of both valine and leucine. Since these
amino-acids are of comparable chemical structure, Glad-
stone concluded that they are synthesized by similar, if not
identical, routes and that the addition of only one of the
acids resulted in the competitive inhibition of one or more
of these routes. Several examples of this type of effect have
been found during experiments with Neurospora mutants
[cf. 45]-

Mutants

Much valuable information concerning the routes of bio-
logical synthesis of natural compounds has been derived by
the use of mutants, i.e. from organisms genetically different
from the parent strains. If this difference results in inability

66 NITROGEN METABOLISM

to synthesize a compound essential for life, the mutant is
unable to grow unless it can obtain from the environment
at least one of the products of the reaction which it is unable
to accomplish. Such a reaction is often referred to colloqui-
ally as a 'genetically blocked reaction'. Beadle and Tatum
predicted that it should be possible to deduce the sequence
of reactions in biosyntheses from the range of compounds
which replace the substances required by nutritionally-
exacting mutants. The organisms used in these studies
include Penicillium notatum, Aspergillus niger. Asp. nidulans,
Ophiostoma, Esch. coli and B. subtilis, but the ascomycetes
Neurospora crassa and N. sitophila still remain the most
suitable if precise genetic data is also required. Most natural
(i.e. 'wild type') strains of Neurospora grow on a simple
medium containing mineral salts, biotin, an inorganic
source of N and an organic source of C and energy (e.g.
sucrose, sorbitol) and must therefore possess the wide
variety of enzymes required for the synthesis of all the
normal constituents of cytoplasm. The vegetative phase
reproduces asexually by conidia and micro-conidia; sexual
reproduction is only possible between gametes from parents
of opposite mating types. Much is known concerning the
genetics of Neurospora [see 5] and since the vegetative phase
is haploid, there are in contrast with diploid organisms, no
problems concerning the dominance of one character over
another. Strains whose nutritional requirements are different
from the parent type may arise naturally by spontaneous
mutation, but such mutations are often few in number and
natural selection does not favour their survival. In order to
increase the chance of isolating such mutants, the mutation
rate is artificially increased by exposing the conidia to ultra-
violet light. X-rays or chemical mutagens, e.g. mustard gas.
The conidia are then transferred to the protoperithecia of
wild type Neurospora of opposite mating type, and in conse-
quence asci develop, each ascus containing eight spores. One
spore from each ascus is transferred to a solid medium con-
taining the minimal requirements for growth (minimal
medium), and after incubation the colonies that have
developed will be of the wild type. Their position is noted

SYNTHESIS OF AMINO-ACIDS 67

and agar containing known additional nutrients is layered
over the original plate. Any new colonies which develop
after further incubation are derived from mutant spores, and
after being subcultured their nutrition can be studied in
more detail. Special techniques may be required in order
to promote the formation of discrete colonies, particularly
with organisms whose growth tends to spread (many strains
of Neurospora). A more laborious method of isolation is to
subculture each ascospore on a rich medium, i.e. one con-
taining amino-acids and gro-wth factors, and then transfer
to a minimal medium. If there is no growth on the latter,
the nutrition of the parent colony is then examined further.
Other more efficient, technically easy and less laborious
methods for selecting mutants of various organisms have
been described [18, 31, 32].

Using such techniques, a number of mutants have been
obtained which are exacting towards a particular amino-
acid, growth factor, purine or pyrimidine. Many appear to
be unable to perform reactions expected to take place in
one step, e.g. the amination of inosine to form adenine, and
Beadle and Tatum have advanced the hypothesis that each
enzyme is controlled by a specific gene, and any change
in the latter is reflected by an alteration in the enzyme's
activity. Although the evidence is indirect and has been
criticized by Delbruck [11] an analysis of the available infor-
mation shows that at least 73% of the genes of Neurospora
have only one function, and there are no indisputable
examples of genes with two or more functions [30]. It must
be stressed that mutation may involve modification rather
than complete loss of the gene and the corresponding
enzyme, e.g. a Neurospora mutant unable to synthesize
tryptophan from indole and serine still possessed the requi-
site condensing enzyme, though in an inactive state [22].
(Cf. also the synthesis of pantothenic acid [51]).

In order to show whether mutants requiring the same
factor are due to mutation of either the same gene or com-
pletely different genes, one of two tests may be applied. In
organisms with a sexual cycle (e.g. Neurospora), each mutant
is mated with a parent of known genetic composition and the

68 NITROGEN METABOLISM

percentage recombination of characters obtained in the pro-
geny of each cross will indicate whether or not the mutants
are the outcome of mutation at the same gene locus. The
cells of fungi are multinucleate and it frequently happens
that hyphae from two parents will fuse and thus form a
structure known as a heterocaryon which contains nuclei
derived from each of the parent hyphae. Consequently a
heterocaryon formed between two genetically identical
mutants will have the same growth requirements as the
two parent mutants. But the heterocaryon from genetically
different mutants will grow in the complete absence of such
compounds since the metabolic deficiencies of the nuclei
derived from one parent will be complemented by the
activities of the nuclei from the other parent.

Primarily because of the ease with which they can be
isolated, mutants of penicillin-sensitive bacteria have been
used in many recent investigations [9]. Mutation is brought
about by irradiation with ultraviolet light in doses sufficient
to kill 99*9% of the organisms and then the suspension is
cultured in a rich medium, washed and transferred to mini-
mal medium containing penicillin. The non-mutants grow
and are consequently killed by the penicillin. The suspen-
sion is then plated on to a rich medium and the colonies
appearing are usually those of nutritionally exacting mutants.

Having isolated a number of mutants exacting towards a
particular substance, other substances likely to be inter-
mediates in its synthesis are then tested for their ability to
promote grov^h, since theoretically the product of the
blocked reaction or any compound derived from it should
be active in this respect, provided the organism is permeable
to such substances. The sequence of intermediates in a
biosynthesis may then be deduced by arranging the mutants
in order according to the range of compounds supporting
grov^h. It is to be expected that the further the blocked
reaction from the end-product, the greater the number of
compounds utilized by the mutant. For example, in the
synthesis of D from A by:

PLATE I. — Syntrophism among arginine requiring mutants of
Esch. coli. Mutant O responds to ornithine, citrulline or
arginine; C to citrulline or arginine; A to arginine only.
Photograph of growth after 48 hr. at 37° C. on a medium
containing suboptimal amounts of required nutrients. Note
the enhanced growth of C and O due to the secretion by A
of a substance (citrulline?) which can be utilized by C and O.
Similarly the enhanced growth of O due to the secretion by
C of a substance (ornithine?) utilized by O [7]

SYNTHESIS OF AMINO- ACIDS 69

the growth of a mutant incapable of reaction a will be sup-
ported by B, Cor D\ if incapable of b, by C or D\ and if in-
capable of c, the mutant will only grow in the presence of D.

For convenience, mutants are described either by adding
the suffix '-less' to the substance required for growth, e.g.
arginineless denotes exacting towards arginine, or by using
the term auxotroph, e.g. an arginine auxotroph.

Growth in the presence of suboptimal amounts of the
product of a blocked reaction may result in the excretion of
the precursor of this reaction into the medium. Consequently
when two related mutants are streaked near one another on
a solid medium containing suboptimal amounts of required

HOOC(cH2)2CH(nH2)COOH NH2CONhCcH2)3Ch(nH2)cOOH

Qlutomic acid \ / citrulline

NH2(cH2)3Ch(nH2)cOOH
^ ornithine

proline

CH,— CH, / rjjHj NH2

CO ^CNH(cH2)3Ch(nH2)COOH

CHj^CHCOOH NM2 UU arginine

NH urgQ

FIG. 5.1. — The arginine cycle and the route of arginine synthesis
in Neurospora crassa and Penicillium notatum

nutrients, one may secrete a substance which is used directly
by the other or changed by it into a form which can be
utilized by the former mutant. The enhanced growth which
results is readily visible and this phenomenon of 'syn-
trophism' has been widely exploited by Davis [7] (Plate I).

Arginine synthesis

Seven genetically distinct arginineless mutants of A^. crassa
were isolated and of these, four grew on ornithine, citruUine
or arginine; two on citrulline or arginine and one on arginine
only. The organism also possessed arginase and urease and
it was concluded that there is an 'arginine cycle' in Neuro-
spora (Fig. 5.1) comparable to that described by Krebs in
mammalian liver. It can be deduced frorn the genetic data
that there are at least four steps in the synthesis of ornithine

70 NITROGEN METABOLISM

and two in the conversion of ornithine to citruUine. In
Hver, the synthesis of citrulUne from ornithine, CO 2 and
NH3 proceeds by a mechanism utiUzing metaboHc energy
and with carbamylglutamic acid as an essential co-factor.
Contrary to expectation, the latter does not function by
transferring the carbamyl group directly to ornithine. In the
presence of ATP and Mg"^"^, the citrulline combines with
aspartic acid and the product subsequently undergoes
hydrolysis to yield arginine and malic acid [38]. Comparable
systems have not yet been described in micro-organisms. In
Lb. arabinosus glutamine appears to play an essential role in
arginine synthesis, the amide group being used in the
formation of citrulline from ornithine [37^].

A mutant of P. notatum grew on arginine, citrulline,
ornithine or proline, whilst another grew on either of these
amino-acids or glutamic acid. A third was known to grow
only on proline, indicating that proline is not itself on the
direct route of arginine synthesis but is probably related
to a precursor of ornithine [3, also cf. 43]. The possible
relationships between glutamic acid, proline and the ar-
ginine cycle are shown in Fig. 5.1. Studies of the nutri-
tion of naturally occurring strains of lactobacilli [50] and
mutants of Esch, coli provided evidence that the mechanism
of arginine synthesis in bacteria is the same as in the fungi.
Using mutants of Esch. coli, Davis and his colleagues [8]
have shown that proline is formed by the reduction of
A^-pyrroline-5-carboxylic acid (PCA), a compound formed
from the y-semialdehyde of glutamic acid (GSA).

CH2 — CH.2 CH2 — Crl2

II II -H,0

HOOC CH.COOH >CHO CH.COOH

/ /

NH2 NH2

Glutamic acid (GSA)

CH2 — Cri2 CHg — Cri2

II +2H I I

CH CH.COOH > CH2 CH.COOH

\/ \/

N NH

(PCA) Proline

SYNTHESIS OF AMINO-ACIDS

71

The relationships of these compounds to ornithine has still
to be fully elucidated [lyb]. The conversion of glutamic
acid to ornithine probably proceeds via N-acetylglutamic
acid — > N-acetylglutamic acid y-semialdehyde — > a-N-
acetylornithine — > ornithine [see id].

Tryptophan and nicotinic acid

The interrelationships betv^een tryptophan, the other
aromatic amino-acids and nicotinic acid, as revealed by
experiments with N. crassa and Esch. coli, are summarized in
Fig. 5.2. Initially, two tryptophanless mutants oi Neurospora
(10575 ^^^ 40008) were isolated, both utilized indole in
place of tryptophan but only one (40008) utilized anthranilic

COCH2Ch(nH2)cOOH

p-amirtobcnzoic acid

phenylolanine

tyrosine

nicotinic ecid

FIG. 5.2. — The tryptophan cycle and the synthesis of nicotinic acid
in Neurospora crassa. Compounds A and B are hypothetical
intermediates, and the details of the relationship between
tryptophan and the other aromatic amino-acids and PAB are
not yet known (see pp. 72-3) [24]
6

72 NITROGEN METABOLISM

acid. Growth of 10575 i^ the presence of limiting amounts of
tryptophan resulted in the appearance in the medium of a
substance supporting the growth of 40008. This material,
presumably the substrate of the blocked reaction, was later
isolated and identified as anthranilic acid. The reaction
sequence is therefore anthranilic acid — >■ indole — > trypto-
phan. The rate of uptake of indole by 10575 was found to
be a function of the concentration of L-serine in the medium,
and after growth had ceased tryptophan was excreted into
the medium. The enzyme forming tryptophan by the con-
densation of serine with indole was studied in cell-free
homogenates of Neurospora mycelia and shown to con-
tain a prosthetic group of pyridoxal phosphate [49]. The
mechanism by which anthranilic acid is converted into
indole remains unknown. By using isotopes it has been
shown that the carboxyl group of the former does not give
rise to any of the carbon in the indole nucleus of trypto-
phan [37«].

A detailed investigation of Neurospora mutants able to
grow on tryptophan or nicotinic acid has confirmed the
conclusion drawn from animal nutrition experiments that
the metabolism of these two compounds is interrelated.
Furthermore, Haskins and Mitchell have proposed that, at
least in Neurospora, there is a 'tryptophan cycle' [24]. By
using the appropriate mutants and growth conditions,
3-hydroxyanthranilic acid, kynurenine and quinolinic acid
have all been isolated from culture filtrates. Like Lb.
arabinosus, the growth of some, but not all, nicotinic acid
auxotrophs of Neurospora is supported by quinolinic acid,
although only in high concentrations [21]. It is therefore
possible that quinolinic acid is a by-product derived from
the substrate of a blocked reaction, rather than a direct
intermediate in the synthesis of nicotinic acid.

Certain mutants of A^. crassa and Esch. colt require tryp-
tophan, phenylalanine, tyrosine and ^-aminobenzoic acid
(PAB), all in large amounts, before there is even slow
growth, indicating that these four compounds may be
derived from a common precursor and that the synthesis
of anthranilic acid and tryptophan is connected with the

SYNTHESIS OF AMINO-ACIDS

73

metabolism of the other aromatic amino-acids. As a result
of a suggestion made by Stanier, Davis found that this
quadruple requirement could be replaced by shikimic acid,
an alicyclic compound known to occur in plants. This acid
has now been isolated from the culture filtrate of a mutant
of Esch. coli and unequivocally characterized. A precursor of
shikimic acid (SKA) has recently been isolated and iden-
tified as 5-dehydroshikimic acid (DSKA) which is in turn
probably derived from 5-dehydroquinic acid (DQA) [8].
Since growth in the presence of the four aromatic acids was
slow and became maximal on the addition of shikimic acid
or filtrates from wild-type cultures, Davis deduced that the
mutants required at least one other aromatic substance, and
one of these has been identified as ^-hydroxybenzoic acid

(DQA)

(POB). Shikimic acid is only utilized by mutants exacting
to at least four aromatic compounds and this multiple
requirement is probably due to the mutation of a single
gene. It is not yet possible to state whether shikimic acid
is in fact a simple precursor of all these aromatic nitrogen
compounds. Davis has suggested [7, 10] that the apparent
complexity in growth requirements is the result of inter-
ference with the synthesis of a key substance which is
responsible for the integration of various parallel and related
pathways of biosynthesis (cf. valine-isoleucine, pp. 65, 76).
Unlike mammals, Neiirospora and Esch. coli cannot convert
phenylalanine to tyrosine.

Cysteine and methionine

Sulphur is found in organic combination in the amino-
acids cysteine and methionine, and most - organisms can
utilize inorganic forms of sulphur at any oxidation level as

74

NITROGEN METABOLISM

a complete source of this element. Mutants exacting towards
various sulphur compounds are the easiest to produce and
isolate. Of four methionineless mutants of A^. crassa, only-
one specifically required methionine, homocysteine was just
as effective for two of the mutants, whilst the other grew on
cysteine, homocysteine or methionine. The culture filtrate

l:

NH2 CH2

COOH CH.NHj
COOH
cystathionine

CHjSH

— yCHj _

CH.NHj

COOH

hornocyitaine

CHjSCHj

(fHjSH

syi

CHjOH

CH.NH,
I ^

COOH

COOH
methionine

r3

CHOH
I
CH.NHj

COOH

(jiHjS.SOaH

CH.NH2

COOH

cysteine-s-iul phonate

homoierine «/- ominobutyric ocid threon in e

. ' ll(

CHOH ». y

CHg^HjCHa
^ C.OH
CHOH

COOH
o(-keto.i3 hydroxybutyric acid

CHjOH HjSjOg^— H^SOjV-HzSOav- H2Sq4

CH.NHj

COOH *—

^>A=1

FIG. 5.3.

HCOOH

CH.NHj
COOH
glycine

-Pathways for the synthesis of cysteine
and isoleucine

COOH
di hydroxy-^- ethyl
butyric ac'i?"

i
CHg^CHjCHa
CH

CH,NH2
COOH

isoleucine

, methionine

of one homocysteine auxotroph was found to contain a sub-
stance capable of supporting the growth of the other homo-
cysteine autotroph and also of the mutant which would
grow on cysteine. This substance was isolated and identified as
cystathionine [29], and the suggested biosynthetic sequence
is shown in Fig. 5.3. Methionine is probably synthesized by
methylation of the homocysteine produced by the cleavage
of cystathionine (see p. 149).

SYNTHESIS OF AMINO- ACIDS 75

A Neurospora mutant requiring threonine grew poorly
unless the medium was also supplemented with methionine,
cystathionine or homocysteine, indicating that all these com-
pounds were derived from a common precursor. The latter
is probably homoserine since this amino-acid can replace
both threonine and the sulphur-containing amino-acids
[46]. Moreover, if homoserine can couple with cysteine the
product would be cystathionine. As regards the incorpora-
tion of inorganic forms of sulphur into organic compounds,
it has been suggested that this proceeds via the synthesis of
cysteine by cysteine desulphurase, an enzyme known to
decompose cysteine into HgS, NH3 and pyruvic acid.
There is, however, no evidence that the enzyme can catalyse
the reverse reaction. In Asp, ntger, Asp. nidulans and P.
Tiotatum [27], thiosulphate, and not sulphide, may be an
important intermediate in the synthesis of cysteine. The
sulphur requirements of thiosulphate auxotrophs of Asp.
7iidulans were satisfied by cysteine- S-sulphonate (a thiosul-
phate derivative of serine) and growth was extremely luxuri-
ous in the presence of serine and thiosulphate [28]. Hocken-
huU therefore proposed that the route of sulphur utilization
involves reduction of sulphate to sulphite which is then
converted to thiosulphate, perhaps with the intermediate
formation of sulphoxylate, and that finally thiosulphate is
condensed with serine or some other Cg-compound. A
different sequence has been proposed for Ophiostoma multi-
annulatum [see 5] and N. crassa:

Cs-compound (alanine?) plus S07~ — >- cysteic acid — >

cysteine sulphinic acid — >- cysteine

Lysine and threonine

The route of lysine synthesis in Neurospora appears to be
different from that in Esch. coli. Lysine auxotrophs oi Neuro-
spora were able to utilize a-aminoadipic or e-hydroxy-
a-aminocaproic acid but not a-ketoadipic, a,a'-diamino-
adipic or a,£-diaminopimelic acid. The biosynthetic se-
quence in N. crassa is believed to be:

a-aminoadipic acid — >-

€-hydroxy-a-aminocaproic acid — >■ — > — > lysine [20]

^6 NITROGEN METABOLISM

Unlike Neurospora, Esch. coli contains a,e-diaminopimelic
acid (DAP) and possesses a specific enzyme converting it
into lysine and CO 2 (p. 29). This decarboxylase is not
present in those lysineless mutants which accumulate large
amounts of DAP in their culture media. The lysine require-
ments of other mutants were satisfied by DAP but not by
a-aminoadipic or a-amino-£-hydroxycaproic acid, and the
conclusion was reached that DAP is the immediate pre-
cursor of lysine in Esch. coli. Moreover, DAP and threonine
may be derived from a common precursor, and a-amino-
butyric acid may be an intermediate in the synthesis of
threonine from homoserine (Fig. 5.3) [9].

Valine, isoleucine and threonine

A mutant (161 17) of N. crassa would only grow when
provided with both L-valine and L-isoleucine, yet as far as
could be ascertained it diifered in only one gene from the
wild type parent. For optimal growth, the ratio of L-valine
to L-isoleucine was critical (7:3) and increasing the concen-
tration of either acid adversely affected growth. The a-keto
acids corresponding to valine and isoleucine ('ketovaline'
and 'ketoisoleucine') supported the growth of other mutants,
and 161 17 would grow in the presence of isoleucine and
'ketovaline' but not valine and 'ketoisoleucine'. Moreover,
'ketoisoleucine' inhibited the growth of another mutant
requiring only 'ketovaline'. Bonner has suggested that
16117 is unable to synthesize isoleucine and that the sub-
strate of the blocked reaction ('ketoisoleucine'?) accumulates
and competitively inhibits the synthesis of valine (from
'ketovaline'?), with the result that the mutation of one gene
appears to bring about the blocking of two reactions [3].
After 161 17 has grown in the presence of isoleucine and
valine, the medium contains a,^-dihydroxy-^-ethylbutyric
acid, a substance replacing isoleucine for an auxotroph of
Esch. coli; no 'ketoisoleucine' could be detected [ib]. Iso-
leucine auxotrophs of N. crassa, B. suhtilis and Esch. coli
can be divided into three groups according to the com-
pounds which they can utilize: (i) only isoleucine, (2)
a,j5-dihydroxy-/5-ethylbutyric acid or isoleucine, (3) iso-

SYNTHESIS OF AMINO-ACIDS 77

leucine, a,y5-dihydroxy-/?-ethylbutyric acid, a-aminobutyric
acid, a-ketobutyric acid or threonine. Hence, the metabo-
lism of isoleucine, the sulphur amino-acids and threonine
is closely related and may proceed from a common C4-
precursor. Isoleucine may arise from the latter by the
addition and reduction of an acetyl group [ib] (see Fig. 5.3).
The keto-acids of valine and isoleucine have been identified
in the culture filtrate of an isoleucinless mutant of Esch.
coli [47].

Isotopes

Isotopes of carbon are proving useful in tracing the
origin of the various carbon atoms of amino-acids, and their
application to the study of syntheses in micro-organisms
(Torulopsis utilis, Esch. coli, N. crassa) is mainly due to
Ehrensvard and his colleagues [6, 12, 13]. By using acetate
as a sole source of carbon and labelling the carbon in the
two positions with different isotopes (C^^HgC^^OOH), it is
possible to determine whether a particular C atom is derived
more or less directly from the CH3 or the COOH group.
With the yeast T. utilis adapted to growth on acetate, it was
found that two acetate carboxyl groups were liberated as
respiratory CO 2 for every methyl group. After hydro-
lysing the yeast with acid, the amino-acids were isolated
by means of electro-dialysis and chromatography on ion
exchange resins. The ratio of C^* to C^^ in the carboxyls
of most of the amino-acids was the same as that in the
respiratory CO 2 , thus demonstrating that CO 2 fixation had
taken place. In general, all the alpha-C atoms and many of
those in the side chains were derived from the methyl group
of the acetate. It was concluded that glutamic acid is a
precursor of arginine (cf. p. 70) and that lysine is synthe-
sized by the head to tail condensation of acetyl radicals.
When NHa-CHgC^^OOH was used as a sole N source the
C^* was finally located mainly in the glycine, serine and
proline of the proteins. Thus in Torulopsis, as in bacteria
(p. 149) and animals [cf. 14], glycine is a precursor of serine.
By growing cultures of Sac. cerevisiae, T. utilis and Ps.
fluorescens in the presence of HC^*OOH, it has been shown

78 NITROGEN METABOLISM

that the C in position 2 of the imidazole ring of histidine
is derived exclusively from formate [33, 44].

Isotopically labelled compounds such as amino-acids can
be readily prepared by isolating them from organisms which
have been grown in media containing substances enriched
with the appropriate isotope [2]. Non-exacting organisms
are especially useful since they utilize simple substances
(HC07,NHt, SOI") as sources of C, Nand S and such
substances enriched with the appropriate isotopes are
readily available.

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\h. Aldelberg, E. A. (1951), J. Bact., 61, 365
\c. Altenbern, R. A. and Housewright, R. D. (1953), J. hiol.
Chem., 204, 159

2. Aquist, S. E. (195 1), Acta chem. Scand., 5, 103 1

3. Bonner, D. (1946), Cold Spr. Harb. Symp., 11, 14

4. Braunstein, A. E. and Kritzmann, M. G. (1937), Enzymologia,

2, 129

5. Catcheside, D. G. (195 1), The Genetics of Micro-organisms

(Pitman, London)

6. Cutinelli, C, Ehrensvard, G., Reio, L., Saluste, E. and

Stjernholm, R. (195 1), Acta chem. Scand., 5, 353

7. Davis, B. D. (1950), Experientia, 6, 41

8. (1952), Intermediates in the Biosynthesis of Proline, etc.,

in Biological Systems, 2nd Internat. Congress of Biocheni.

9. (1952), Nature, 169, 534

10. (1952), J. Bact., 64, 729, 749

11. Delbruck, M. (1946), Cold Spr. Harb. Symp., 11, 22

12. Ehrensvard, G. (1952), On the Origin of Aromatic Structures

in Biological Systems, 2nd Internat. Congress of Biochem.

13. Reio, L., Saluste, E. and Stjernholm, R. (1951), J. hiol.

Chem., 189, 93

14. Feldman, L. I. and Gunsalus, I. C. (1950),^. biol. Chem., 187,

821

15. Fildes, P. (1945), Brit. J. exp. Path., 26, 416

16. and Rydon, H. N. (1947), Brit. J. exp. Path., 28, 211

i7<3. Fincham, J. R. S. (1950), J. biol. Chem., 182, 61

176. (1953), Biochem. J., 53, 313

18. Fries, N. (1947), Nature, 159, 199

19. Gladstone, G. P. (1939), Brit. J. exp. Path., 20, 189

20. Good, N., Heilbronner, R. and Mitchell, H. K. (1950), Arch.

Biochem., 28, 464

21. Gordon, M., Haskins, F. A. and Mitchell, H. K. (1950), Proc.

Nat. Acad. Set., Wash., 36, 427

SYNTHESIS OF AMINO- ACIDS 79

22. Gordon, M. and Mitchell, H. K. (1950), Genetics, 35, no

23. Green, D. E., Leloir, L. F. and Nocito, V. (1945), J. biol.

Chem., 161, 559

24. Haskins, F. A. and Mitchell, H. K. (1949), Proc. Nat. Acad.

Sci., 35, 500

25. Herbst, R. M. (1944), Advances in Enzymology, 4, 75

26. Hird, F. R. J. and Rowsell, E. V. (1950), Nature, 166, 517

27. Hockenhull, D. J. D. (1948), Biochem. J., 43, 498

28. (1949), Biochem. Biophys. Acta, 3, 326

29. Horowitz, N. H. (1947), J. biol. Chem., 171, 255

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31. Lederberg, J. and Tatum, E. L. (1946), J^. biol. Chem., 165, 381

32. Lein, J., Mitchell, H. K. and Houlahan, M. B. (1948), Proc.

Nat. Acad. Set., 34, 435

33. Levy, L. and Coon, M. J. (1951), jf. biol. Chem., 192, 807

34. Lichstein, H. C. and Cohen, P. P. (1945), J. biol. Chem., 157,

35. Gunsalus, I. C. and Umbreit, W. W. (1945), jf. biol.

Chem., 161, 311

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366. Sober, H. A. and Peterson, E. A. {1952), J. Amer. chem.

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37a. Nye, J. F., Mitchell, H. K., Leifer, E. and Langham, W. H.

(1949), J. biol. Chem., 179, 783
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Chem., 207, 267
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amino-acids in protein synthesis in yeast
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40. Rydon, H. N. (1948), Brit. J. exp. Path., 29, 48

41. Shemin, D. (1949), Cold Spr. Harb. Symp., 14, 161

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165,731

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51. Wagner, R. P. (1949), Proc. Nat. Acad. Sci., 35, 185

CHAPTER VI

ABSORPTION OF AMINO-ACIDS BY
MICRO-ORGANISMS

The amino-acids required for growth are either synthesized
by the organism itself or derived from its environment.
Consequently the mechanisms controlling the absorption of
amino-acids are of fundamental importance, especially to
exacting organisms who must perforce rely entirely on their
environment for supplies of compounds which they are un-
able to synthesize. The factors influencing the passage of
amino-acids into and out of micro-organisms, particularly
those exacting towards amino-acids, have been investigated
by Gale and his colleagues [6]. Whilst using the bacterial
decarboxylases [4] (p. 27) for the analysis of the amino-acid
composition of bacterial proteins [3], Gale found that
Gram-positive bacteria contained large amounts of free
amino-acids. This discovery arose from the fact that hydro-
lysates of whole cells, and not isolated proteins, were being
analysed, and it was realized that serious errors would result
if there were appreciable amounts of free amino-acids in
the experimental material prior to hydrolysis. Such amino-
acids might be adsorbed on to the cell surface or be present
inside the cells. In order to determine the significance of
these possibilities, washed cells of Strep, faecalis were dis-
integrated by shaking with glass beads, a procedure which
ruptured the cells but caused no significant degradation of
the proteins. Analysis of the disintegrated cells revealed the
presence of relatively high concentrations of certain amino-
acids (cf. columns h and a—hm Table 6.1). Although the
latter were also found in disintegrated cells of Staph, aureus,
there were no significant amounts in those of two Gram-
negative organisms, Esch. colt and Aerobacter aerogenes [5].
Further experiments showed that the greater proportion of
these amino-acids was located inside the cells.

There was therefore an indication that only Gram-
80

ABSORPTION OF AMINO-ACIDS 8l

positive bacteria contained free amino-acids. Moreover,
since many amino-acid exacting organisms are also Gram-
positive, their presence might be the outcome of mechanisms
enabUng such organisms to absorb essential amino-acids

TABLE

6.1

THE AMINO-ACID COMPOSITION OF STREP. FAECALIS

CELLS [5;

Results expressed

in terms of amino-

acid N as

0/

/o

total N

Amino-acid

Acid
hvdrolysate

Disintegrated
cells
ib)

Combined

amino-acid

{a-b)

L(+)-Lysine
L(+)-Argimne
L(+)-Glutamic acid
L(-)-Histidine
L(+)-Omithine
l(— )-Tyrosine

11-30

5-19
5-60
2-84
1-48
o-8i

2-90
010
1-74
0-70

1-27

o-o

8-40
5-09

3-86

2-14
0-2I

o-8i

from their environment. In view of the potential signifi-
cance of these observations, Gale decided to undertake a
series of more detailed investigations. Apart from the intrinsic
value of the results these experiments provide a valuable
example of one type of approach to microbiological problems.

Procedure for assaying internal amino-acids

A thick suspension (about 30 mg.dry \vt./ml.) of washed
cells of the organism is prepared and the amount of external
amino-acid is determined by adding the appropriate decar-
boxylase preparation to a sample of this suspension. The
total amount of free amino-acid, i.e. the amount inside the
cells plus that outside, is determined by adding the decar-
boxylase to a sample of the suspension which has been
previously heated at 100° C. for 15 minutes in order to dis-
rupt the cells. The amount of amino-acid inside the cells
is then readily calculated by subtracting the former result
from the latter. Concentrations are expressed in terms of
either the amount of amino-acid in a specified dry weight
of cells, or the amount per millilitre of cell volume or
'internal free-space'. The volume occupied by the cells was

82 NITROGEN METABOLISM

obtained by centrifuging samples of the whole cell suspen-
sion in graduated tubes and the volume of the cellular con-
stituents was calculated by assuming that they were mainly
proteins whose specific volume is 0-70. Subtraction of the
volume of the cellular constituents from the volume of an
equivalent amount of whole cells yields a value for the
'internal free-space', i.e. the internal environment [5].

The amino-acid decarboxylases are enzymes of high
specificity (p. 27), consequently this procedure will esti-
mate only amino-acids which are initially free in the cells or
which are liberated from compounds that are easily decom-
posed during the assay procedure, e.g. by heat or by other
enzymes present in the decarboxylase preparations. Whilst
enzymic decomposition during the assay procedure has not
been ruled out, it is unlikely that they arise by the decompo-
sition of heat-labile compounds since the same amount of
amino-acid is found in cells disrupted by heat as in those
made permeable by treatment with tyrocidin or phenol [13].
In the absence of evidence to the contrary the internal
amino-acids are regarded as being 'free' in the sense that
they are chemically uncombined.

Internal amino-acids in Gram-positive organisms

Washed suspensions of twenty-seven organisms com-
prising thirteen genera were prepared from cultures which
had been grown on a medium rich in amino-acids. The
experiments were restricted to the six amino-acids for which
specific decarboxylases were then known. Free amino-acids
w^ere found only in Gram-positive organisms, yeasts as well
as bacteria, and none were detected in Gram-negative
bacteria. The yeasts contained high concentrations of all
six of the amino-acids whereas the bacteria contained only
lysine and glutamic acid in appreciable amounts [18].

Absorption of amino-acids by washed cells

The factors controlling the absorption of lysine and
glutamic acid have been studied in experiments with washed
cell suspensions of Strep, faeca lis and Staph, aureus {Micro-
coccus pyogenes var. aureus), both of which are exacting

ABSORPTION OF AMINO-ACIDS 83

towards amino-acids. Cells which initially contain negligible
amounts of amino-acids (i.e. 'amino-acid deficient cells') are
especially suitable for this type of experiment and they were
obtained from cultures grown on a liquid medium in which
the concentration of amino-acids was just sufficient for
growth. Such cultures were harvested near to the cessation
of active cell division since the ability to absorb amino-
acids was observed to decline appreciably in the stationary
phase. The amino-acid deficient cells were suspended in an
inorganic salt medium (pH 7-2) to which other substances
(amino-acids, glucose, etc.) were added as required. At the
end of the experimental period the cells were collected by
centrifuging, washed, made into a thick suspension and
the internal concentration of the amino-acid determined.
Unless stated to the contrary the results given below apply
to Strep, faecalis.

Uptake of lysine [5]

As soon as lysine was added to the experimental system
it began to pass into amino-acid deficient cells of Strep,
faecalis, and after about 15 minutes equilibrium was reached
and there was no further increase in the internal concen-
tration. This uptake of lysine occurred rapidly at 37° C. and
was still appreciable at 4° C. The rate of appearance of
lysine inside the cells w^as approximately directly propor-
tional to its concentration in the external medium (Fig. 6.1),
and the Q^q over the range of 20°-30° C. was i -4. From these
facts it may be deduced that lysine is entering the cells by
a process of diffusion. However, this uptake did not repre-
sent simple equilibration betw^een lysine-deficient cells and
a lysine-rich environment, since at equilibrium the internal
concentration of the amino-acid was from three to twenty
times greater than the external concentration, the ratio of
internal to external concentration being inversely related
to the latter. In other words, the cells were accumulating
lysine against a concentration gradient (Fig. 6.2). The rate
of lysine absorption increased as the hydrogen-ion concen-
tration of the salt medium was decreased to pH 9-5, and
since the isoelectric point of lysine is 9*47, it is feasible that

84 NITROGEN METABOLISM

lysine most readily enters the cells as the zwitterion. When
the cells fermented glucose, the uptake of lysine was de-
pressed but could be restored, although not completely, if
glutamic acid was also added to the system. The factors
influencing the outward migration of lysine were also of
importance, if only to show that washing the cells prior to

1 1 1

O 5 10 15

EXTERNAL CONG. OF AMINO- ACID (a' mole/mi.)

FIG. 6.1. — Effect of external concentration of L-Iysine (#) and
L-glutamic acid (O) on the rate of entry of the amino-acid
into Strep, faecalis

assay did not remove any of the internal amino-acids. Cells
containing large amounts of lysine, glutamic acid and
probably several other amino-acids were obtained from cul-
tures grown in the presence of a tryptic digest of casein.
When these 'amino-acid rich cells' were incubated in an
amino-acid free salt medium, there was no outward migra-
tion of either lysine or glutamic acid unless the cells were
fermenting glucose.

ABSORPTION OF AMINO- ACIDS 85

More recent experiments have served to emphasize that
the previous history of the cells and the presence of other
amino-acids profoundly influence the inward as v^ell as the
outward migration of lysine and glutamic acid. Cells con-
taining only lysine in large amounts were obtained by

50-1

4-0-

3-0-

«/)2-0-

r20

-16

hi2

d

z
o
o

z
cr

1 £- ^

EXTERNAL CONG. (/* mole/m.l.)

?i

-4.

20

FIG. 6.2. — Effect of external concentration of lysine on (a) internal
concentration of lysine (#) and (b) the ratio of internal con-
centration of lysine to external concentration: in ratio calcu-
lations internal concentrations expressed in terms of volume
of intact cells (A) and volume of intact cells less that of solid
debris (^). Strep, faecalis suspended in lysine solutions for
3 hr. at 4° C. [5]

incubating a washed suspension of amino-acid deficient
cells in the presence of lysine [16]. When these cells were
transferred to an amino-acid free environment (o'i5 M.-NaCl
or Na2HP04 at 37° C.) lysine migrated out into the sur-
rounding medium and, in contrast to the previous experi-
ments with amino-acid rich cells, the migration occurred in

86 NITROGEN METABOLISM

the absence of glucose fermentation. It may be concluded
that the presence of high concentrations of other amino-acids
in the internal environment promotes the retention of lysine
inside the cells and that their influence is overcome by
glucose fermentation. The converse is also true, since the
presence of acidic or basic amino-acids in the external
environment retarded the uptake of lysine by amino-acid
deficient cells in the absence of glucose. The effects of
orthophosphate on the outw^ard migration of lysine have
been investigated [i6], but the results are difficult to inter-
pret and the reader is referred to the original paper.

From all these results, it may be deduced that the accumu-
lation of lysine by Strep, faecalis involves a diffusion mech-
anism which is independent of metabolic energy and may
be due to the establishment of a Donnan type of equilibrium.
Such a hypothesis is supported by the observation that,
when suspended in an amino-acid free medium, lysine
migrated out of cells which contained only this amino-acid
in large amounts. However, it must not be assumed that the
mechanism of lysine absorption is the same in all Gram-
positive organisms. Although similar results were obtained
with Staph, aureus, the uptake of lysine by Sac. cerevisiae
was dependent on metabolic energy [6] and is therefore
comparable to the uptake of glutamic acid by Strep, faecalis
and Staph, aureus.

Uptake of glutamic acid [5]

Irrespective of the pH of the inorganic salt medium there
was no absorption of glutamic acid by the amino-acid de-
ficient cells unless glucose or a tryptic digest of casein was
also added. Hence, unlike lysine, the uptake of glutamic
acid appeared to be an endergonic process utilizing meta-
bolic energy made available by the fermentation of glucose
or by the metabolism of amino-acids. Arginine promoted
the absorption of glutamic acid, although not as efficiently
as glucose, and the fact that the organisms possess arginine
dihydrolase (p. 27) may be of some significance. In the
presence of glucose, glutamic acid was not only absorbed
but also concentrated in the cells, and the relationships

ABSORPTION OF AMINO-ACIDS 87

between the internal and external concentrations at equili-
brium were similar to those found for lysine (cf. Fig. 6.2).
But, in contrast to lysine, the rate of uptake was not directly
proportional to the external concentration (Fig. 6.1) and
the relationship was reminiscent of that between the rate
of an enzyme reaction and the concentration of the sub-
strate. The Qjo over the range 2o°-30° C. w^as 1-94, which
is close to 2-0 and therefore indicative of a chemical and
presumably of an enzymic reaction.

Various inhibitors of intermediary metabolism, such as
cyanide and iodoacetate, have no effect on the uptake of
lysine, but any substance inhibiting fermentation also
inhibits the uptake of glutamic acid. However, it is possible
to separate the processes of energy production from those
of energy utilization by preferential inhibition of the latter.
This may be accomplished by using a substance such as
8-hydroxyquinoline which in low concentrations inhibits
the uptake of glutamic acid without affecting fermentation,
although higher concentrations inhibit the latter as well [7].
Since 8-hydroxyquinoline is a chelating agent, the conclu-
sion was drawn that cations played an important role in
glutamic acid absorption. Staphylococcus aureus was used in
these experiments and the problem was further investigated
by growing the organism in the amino-acid poor medium
from which one or more cations had been removed. The
ability to absorb and concentrate glutamic acid was seriously
impaired only in cells harvested from media deficient in
Mg"^"^ or Mn"*""^. Whilst there is insufficient data for
deciding which of these ions is the natural activator of the
glutamic acid absorption mechanism, it is pertinent to note
that both of these cations are frequently found as co-factors
of enzymes associated with phosphorylation.

In view of the dependence of the process on metabolic
energy it is conceivable that glutamic acid passes through
the cell wall in the form of a compound whose synthesis is
endergonic (e.g. as glutamine, glutathione, or a phosphory-
lated derivative), and having passed through the cell wall,
this compound is reconverted to the free acid. Glutamine,
glutathione and glutamylglutamic acid all failed to enter
7

58 NITROGEN METABOLISM

the cells unless glucose was also added to the experimen-
tal system. On the other hand, a,}/-diethylglutamate and
N-phosphorylglutamic acid were absorbed in the absence
of glucose and gave rise to internal glutamic acid [8]. Hence
it is possible that glutamic acid passes through the cell wall
in a phosphorylated form, and irrespective of whether the
phosphate group is on the amino group or on one or both
of the carboxyl groups, the synthesis of such a compound
would be endergonic. But, the evidence in favour of this
hypothesis is far from conclusive. Many cells are known to
be impermeable to phosphorylated compounds and the Q^q
for the uptake of N-phosphorylglutamic acid was inter-
mediate between that for free diffusion and for an enzymic
process.

When amino-acid deficient cells of Staph, aureus were
incubated with glutamic acid and glucose together with
mixtures of other amino-acids, the accumulation of free
glutamic acid was reduced and sometimes ceased. This was
the outcome of a marked reduction in the amount absorbed
and also an increase in the cellular combined glutamic acid.
If the concentration of glutamic acid in the external medium
was very much greater than that of the other amino-acids,
assimilation into cell substance was suppressed and some
accumulation of the free acid did take place. Evidently
whether glutamic acid entered the cells and accumulated as
the free acid or whether it was converted into a combined
form, depended on the ratio of glutamic acid to other amino-
acids in the external medium. The presence of single acids
such as aspartic, cysteine, glycine, serine and alanine pro-
duced a marked decrease in the rate of glutamic acid accu-
mulation. Valine, leucine and isoleucine caused a small
increase in the latter, whilst all the other amino-acids
examined had no effect. Aspartic acid acted as a competitive
inhibitor, but this explanation did not apply to cysteine and
alanine, the addition of which led to the synthesis of extra-
cellular peptides containing glutamic acid [lo, ii].

There was a small leakage of glutamic acid when cells of
Staph, aureus containing large amounts of several amino-
acids were incubated in an amino-acid free medium: the

ABSORPTION OF AMINO-ACIDS 89

addition of glucose completely suppressed this outward
migration. On the other hand, there was no outward migra-
tion of glutamic acid from Strep, faecalis unless glucose was
present [5].

The uptake of histidine and aspartic acid by Strep, faecalis
is also dependent on metabolic energy [5] and in Sac.
cerevisiae this applied to all of the amino-acids investigated
[19, 6].

Mechanism of amino-acid absorption

Whilst there are unaccountable differences even between
somewhat similar organisms such as Strep, faecalis and
Staph, aureus (cf. the action of inhibitors in 6, 9), there is
sufficient data to warrant a discussion of the mechanisms
which may be operative in the absorption of amino-acids.
This topic is of particular interest since the physical and
chemical structure of the barrier separating the interior of the
bacterial cell from the external environment is now being
actively studied by several workers [15, 17, 21]. Moreover,
amino-acids exist in solution as ions, consequently their
mode of absorption may be only one aspect of the general
problem of ion transport across cellular membranes [cf. 20].

The normal electrochemical properties of a cell will be
markedly altered by the absorption of ions of one species
unless there is simultaneously an equivalent migration of
ions either of opposite charge into the cell or of like charge
out of the cell. Many experiments with plant and animal
cells show that the uptake of one type of ion usually involves
the concurrent transport of other ions, and it is therefore
legitimate to expect that the uptake of amino-acids is like-
wise associated with the movement of other ions. Whether
this does in fact happen has not yet been investigated, and
indeed little is known about the migration of ions into and
out of micro-organisms. Several plant and animal cells con-
tain higher concentrations of particular inorganic ions than
the extracellular fluids, e.g. the K"^ content of mammalian
cells is greater than that of the plasma. One explanation of
the unequal distribution of diffusible ions between cells and
their environment has been advanced by Donnan, who

90 NITROGEN METABOLISM

proposed that such a distribution in a system in equilibrium
is the natural outcome of the presence of intracellular non-
diffusible ionic substances such as proteins [2].

The experiments with Strep, faecalis, Staph, aureus and
Sac. cerevisiae have shown that certain amino-acids can
pass into the organisms against the concentration gradient
and at equilibrium their internal concentration may be, and
often is, greater than their concentration in the external
environment. Furthermore, two distinct mechanisms appear
to be operating in the absorption of amino-acids, one
involving simple diffusion, e.g. the uptake of lysine by
Strep, faecalis and Staph, aureus, whilst the other involves
the utilization of metabolic energy, e.g. the uptake of glu-
tamic acid. These facts may be accounted for by one of four
explanations:

(i) That as a result of electrostatic attraction, the amino-
acid becomes associated inside the cell with a non-
diffusible ion of opposite charge, thus establishing
a type of Donnan equilibrium [2].

(ii) That once inside the cell, the amino-acid is con-
verted into a compound which cannot itself pass
through the cell wall.

(iii) That the cell is permeable, not to the amino-acid
itself, but to a derivative whose synthesis is
endergonic.

(iv) That the migration of the amino-acid results from
the movement of another ion whose formation or
transport is endergonic.

Mechanism of lysine absorption

Najjar and Gale suggested that the absorption and accu-
mulation of lysine in Strep, faecalis is due to the establish-
ment of a type of Donnan equilibrium [16]. In physio-
logical conditions of pH lysine carries an overall positive
charge, consequently if only lysine is absorbed it is reason-
able to suggest that this is accompanied either by the excre-
tion of an equivalent amount of another cation such as H
or K"*", or by the absorption of an equivalent amount of

ABSORPTION OF AMINO-ACIDS 9I

anion. Even though the latter takes place, there may be
another mechanism which excretes an equivalent amount of
either the same or a different anion together with cations to
replace the lysine absorbed. It may be recalled that fer-
menting yeast absorbs K"^ from a medium containing KCl
and replaces it by H"^ [see 20]; there is apparently no uptake
of Cl~. A possible explanation here is that metabolic energy
is used to form H"^, which is then secreted into the medium
in exchange for K"^, thus making it appear that the uptake
of K"^ is an active process, i.e. dependent on metabolic
energy. It may therefore be suggested that lysine is absorbed
by Strep, faecalis in exchange for cellular K"^ or another
cation which does not have to be formed at the expense of
metabolic energy. However, recent work dealing with the
accumulation of lysine showed that in Strep, faecalis it was
accompanied by a gain in cellular K^ (with no significant
change in cellular Na"*") whereas with Staph, aureus it had
no apparent effect on either the K"^ or Na"*" content of the
cells. In Saccharomyces fragilis, the uptake of lysine was
dependent on glucose fermentation and was accompanied by
the loss of Na"^ and K"*" from the cells [ib] (in none of these
experiments was the migration of 0H~ and H studied).

Mechanism of glutamic acid absorptio?i

Having regard to the second explanation (ii) advanced
above, it might be suggested that the 'free glutamic acid'
of the cell is in the form of glutamine, a substance whose
synthesis is endergonic and which is assayed by the decar-
boxylase preparation as though it is the free acid: moreover,
as glutamine cannot freely diffuse into cells, it is possible
that it cannot itself pass through the cell wall [5]. But only
a small part of the glutamic acid of the internal environment
of streptococci is in fact in this form [14]. Hence the energy
associated with the uptake of glutamic acid may be used in
the manner outlined in the last two explanations, iii and iv.
The former of these proposes that active transport, i.e. trans-
port dependent on metabolic energy, involves chemical

92 NITROGEN METABOLISM

reactions between cellular constituents and the substance
being transported across the cell membrane [20]. This
implies that the latter contains a substance or carrier which
reacts with the transported substance to form a relatively
stable product which traverses the cell membrane and then
either undergoes chemical decomposition or takes part in an
exchange reaction, thus liberating the transported substance
into the internal environment. Energy may be required for
the formation of either the compound traversing the mem-
brane or the substance which takes part in the final exchange
reaction. Davies and Krebs [id] have shown theoretically
how metabolic energy may be utilized for the production of
H"^ or 0H~ from water. By analogy with their hypotheses
concerning ion transfer in brain cells it may be proposed
that metabolic energy is used to form an excess of 0H~>
which may perhaps combine with CO 2 to form HCO 3", and
that the bacterial cell wall contains a basic ion exchange
complex (X). If the cell contains a high concentration of an
anion such as 0H~ or HCO^ and the external environment
contains glutamate ions, and if it is assumed that the ion
exchange complex X can move in the cell wall and thus
come into contact with the external and internal environ-
ments, the following reactions can be expected to take place,
the equilibrium being towards the right:

internally: X+ + OH" ^ X-OH

externally: X-OH + glutamate ^ X-glutamate + 0H~

internally: X-glutamate + 0H~ ^ X-OH + glutamate

Glutamate ions will thus be transported into the cell and
will be replaced in the external environment by hydroxyl
ions. Eventually a steady state will be established in which
exchange is still taking place, but there is no further overall
increase in the internal concentration of glutamic acid.
Specificity in ion transport may be due to different ions
being transported by diiferent ion-exchange complexes
whose specificity properties are comparable with enzymes.
The observation by Britten that intracellular glutamic

ABSORPTION OF AMINO-ACIDS 93

acid will exchange with extracellular isotopically labelled
glutamic acid in the absence of metabolic energy can be
explained if there is a carrier mechanism of this type in the
cell membrane. All these theories are purely speculative and
their acceptance or rejection awaits the results of further
experiments. One fact of which account must now be taken
is that the accumulation of glutamic acid within ferment-
ing cells of Staph, aureus, Strep, faecalis and Sac. fragilis is
accompanied by an increase in cellular K"^, the increase
appearing to be of the order i gram atom K'^/mole glutamic
acid [lb].

The full significance of the ability to absorb and accumu-
late amino-acids still awaits complete evaluation since only
three organisms and a restricted number of amino-acids
have so far been investigated. It may be remarked that a
decrease in the nutritional requirements of Staph, aureus is
accompanied by a decreased ability to accumulate amino-
acids [12], but it must also be noted that Sac. cerevisiae is
not exacting towards amino-acids and yet accumulates many
of these compounds [19]. Moreover, washed suspensions of
a large variety of organisms, Gram-positive as well as Gram-
negative, decompose several amino-acids, the implication
being that unless the appropriate enzyme is in the cell
surface the amino-acid enters the cell by free diffusion.

REFERENCES

la. Davies, R. E. and Krebs, H. A. (1952), Biochem. Soc. S.ymp.,

8, Chap. 6
lb. Davies, R., Folkes, J. P., Gale, E. F. and Bigger, L. C. (1953),

Biochem. y., 54, 430

2. Donnan, F. G. (1924), Chem. Rev., i, 73

3. Freeland, J. G. and Gale, E. F. (1947), Biochem. J., 41, 135

4. Gale, E. F. (1947), Biochem. J., 41, vii

5. (1947), J- gen. Microbiol, i, 53

6. (1948), Bull. Johns Hopkins Hosp., 83, 119

7. (1949), J' gen. Microbiol, 3, 369

8. (1950), y. gen. Microbiol, 4, v

9. (195 1), Biochem. y., 48, 286

10. (195 1), Biochem. y., 48, 290

11. and van Halteren, M. B. (1951), Biochem. y., 50, 34

94 NITROGEN METABOLISM

12. Gale, E. F. and Rodwell, A. W. (ig^g), jf. gen. Microbiol., 3, 127

13. and Taylor, E. S. (1947), J. gen. Microbiol., i, 77

14. Mcllwain, H., Roper, J. A. and Hughes, D. E. (1948),

Biochem. J., 42, 492

15. Mitchell, P. and Moyle, J. (195 1), J. gen. Microbiol, 5, 966,

981

16. Najjar, V. A. and Gale, E. F. (1950), Biochem. J., 46, 91
17 Salton, M. R. J. (1952), Biochim. Biophys. Acta, 8, 510

and Home, R. W. (195 1), Biochim. Biophys. Acta, 7, 19,

177

18. Taylor, E. S. (1947), J. gen. Microbiol, i, 86

19. (1949), y. gen. Microbiol, 3, 211

20. Ussing, H. H. (1949), Physiol. Rev., 29, 127

21. The Nature of the Bacterial Surface: Soc. Gen. Microbiol.

Symp., ed. Miles, A. A. and Pirie, N. W., Blackwell,
Oxford, England

CHAPTER VII

PEPTIDES AND PROTEINS

Modern concepts of protein structure are founded mainly
on the results obtained by subjecting proteins of animal ori-
gin to procedures involving partial and complete hydrolysis,
amino-acid analysis, ultracentrifugation, electrophoresis and
X-ray diffraction analysis. From the limited data available
there is, however, no reason to believe that the structure and
general physical properties of the proteins of micro-organ-
isms are in any way different from those of animals and
plants. A few representative microbial proteins have been
purified and in some cases crystallized, e.g. the enzymes
alcohol dehydrogenase, catalase, amylase and an extracellular
proteinase from Sac. cerevisiae, M. lysodeiktiais, B. suhtilis
and Strep, haemolyticus respectively and the toxins of CI.
botulinum and CI. tetani. Analyses of acid hydrolysates of
such proteins and of whole cells by classical precipitation
procedures, microbiological assay [41], the bacterial amino-
acid decarboxylases (p. 27), and chromatography on ion
exchange resins or paper have provided adequate evidence
that the proteins of micro-organisms are composed of the
L-stereoisomers of the same a-amino-acids as are those of
more complex multicellular organisms.

Whilst there is no conclusive proof that D-amino-acids are
constituents of proteins and it is probable that any found in
acid or alkaline hydrolysates have arisen by racemization,
they are not metabolically inert, and indeed several of them
can be utilized by many micro-organisms [36]. The first step
in their metabolism may involve deamination by a D-amino-
acid oxidase (p. 11): alternatively, direct conversion to the
corresponding L-isomer may be accomplished by a racemase,
and at the present time two are known, specific for alanine
[46] and glutamic acid [31] respectively. Both possess a pros-
thetic group of py ridoxal phosphate and the alanine racemase

95

g6 NITROGEN METABOLISM

is widely distributed [46]. Several bacterial peptides, especi-
ally those secreted into the environment, contain D-amino-
acids and, in addition, amino-acids not yet found in peptide
combination in animals and plants (Table 7.1). Whether
these unusual amino-acids are also present in bacterial pro-
teins remains to be proved. On the basis of their biological
activity, the peptides associated with the metabolism of
micro-organisms can be arranged into three groups:

(i) Peptides which are or may be co-factors in inter-
mediary metabolism, e.g. glutathione, the folic acid
factors, biocytin (p. 24), glutamine and aspara-
gine. Though the latter three compounds are not
true peptides, they can be regarded as containing
the 'peptidic bond', — CO — NH— [25].

(ii) Peptides which serve as a source of amino-acids
essential for growth. In natural environments,
these peptides arise as the result of autolysis and
the action of extracellular proteases (pp. 11 5-1 6).

(iii) Extracellular peptides. Among the bacteria, members
of the Bacillaceae are especially active in the forma-
tion of this type of peptide, the majority of which
possess antibiotic activity, an exception being the
polypeptides of D-glutamic acid which may be
attached to the organism in the form of a capsule
{B. anthracis and B. mesentericus) or free in the
medium {B. suhtilis and B. mesentericus) [2]. The
capsular material of B. anthracis consists of chains
of glutamic acid residues linked by gamma peptide
bonds together with chains linked by alpha peptide
bonds. Such a capsule may confer immunity from
attack by proteases of the infected host [15].

Co-factors containing peptide bonds

The available evidence indicates that glutamine and as-
paragine play important roles in intermediary metabolism
and it is to be noted that several co-factors are known which,
like glutamine, contain the y-glutamyl radical. Glutamine
may be essential for initiating the growth of certain organ-
isms, but in many instances it is replaceable by glutamic

<

z >

o .t;

> o

.iri a
•J "-l-l

-S ?• »

.s !->

g

Is
o o

^'^

Ti • -
c t5

sill

S-g 8 8 "g,

&.. aa«-2

o a

Be

08 O

X *' X r 4)

r; O U iJ a
^ (J o ^ —'

•^^ I II

+ +

00

Q C

,^ <U *^

^ .t: •-
3 c S

.S :^

i2 1 o

c; ° H

g 22 c«

.B "^

•r O iS

a-S

■t=Oc

= "- la g..

°^:^'8^.§|:B|

» o ^TS ^.t;

.Sfi-5

. u .^

:H t^ a^ 5^

a, -^

^ at: a2 au Uxw

a a a c a^ 2 5i -^

O O O 3 <U

a a a c a

I I

O O

o o o

+

o

=5 -:^

9 R -2

>• TJ

I I

o o

=2 >>

: <^ a

= s 2

<3 a
Dqoq

Is

V (3

CO CO

'^ ^ (^

O^
O ?

O 4>

.2 S !=

C/2

c
^ S

is

^ U3 C

1 1

.2 S

98 NITROGEN METABOLISM

acid, though usually only if much larger amounts are sup-
plied. When small inocula are used, the glutamine require-
ments of Strep, haemolyticus (groups A and C) are absolute
and cannot be replaced in this manner [10]. Streptococci
decompose glutamine to glutamic acid and ammonia only
when they are fermenting glucose, and the presence of small
amounts of glutamine was observed to stimulate the fer-
mentation of glucose by washed cell suspensions of all the
streptococci examined, irrespective of their glutamine re-
quirements during growth [27]. This stimulation was far
greater than that produced by an equivalent amount of
ammonium glutamate, and the more dilute the suspension,
the greater the stimulation. Glutamine here appears to func-
tion by restoring the intracellular concentration of a dif-
fusible co-factor to an optimal value. Similar results were
later obtained with Ln. mesenteroides, Lb. arabinosus [45] and
CI. tetani [24]. Unlike the streptococci, Pr. morganii [26],
Esch. colt, and CI. welchii [18] are able to hydrolyse the amide
group of glutamine in the absence of glucose fermentation.
Asparaginase — the enzyme system catalysing the hydrolysis
of the amide group of asparagine — is widely distributed in
fungi, yeasts and bacteria [cf. 50], and for this reason aspara-
gine is frequently incorporated in media as a convenient
source of readily available carbon and nitrogen. Asparaginase
has been found, for example, in autolysates of P^. pyocyanea,
Esch. colt, B. siibtiUs and Pr. vulgaris. Asparagine is an
essential nutrilite for some strains of Ln. mesenteroides and
Strep, lactis.

Glutamic acid is also a constituent of the folic acid factors,
substances essential for the growth of Strep, faecalis R and
Lb. casei and of key importance in the metabolism of all
organisms. These factors contain a pterin linked to ^-amino-
benzoic acid (PAB) which is in turn coupled through the
amino group to one or more residues of glutamic acid (one
in synthetic folic acid, three in the fermentation Lb. casei
factor and seven in vitamin B^ conjugate). The linkages be-
tween PAB and glutamic acid and between the various glut-
amic acid molecules probably involve the y-carboxyl groups
of the amino-acid. Whilst the enzyme systems in which folic

PEPTIDES AND PROTEINS 99

acid participates have yet to be isolated, there are good
reasons for beUeving that it is impUcated in the synthesis
ofcertainamino-acids, purines and pyrimidines (pp. 146-51).
Glutathione (GSH), the first peptide to be assigned the
function of a co-factor in intermediary metabolism, was dis-
covered and isolated by Hopkins from yeast and various
animal tissues. Little is known about the distribution of this
peptide in bacteria [4] and its isolation from these organisms
has not yet been reported. The chemical synthesis of GSH
by Harington and Mead provided conclusive proof that it
was y-glutamylcysteinylglycine. Ever since GSH was known
to contain a thiol group, it has been postulated that GSH
entered into cellular oxido-reduction reactions:

2GSH —^ OS— SG + 2H

In the presence of glutathione reductase, an enzyme found
in yeast, plants and animals, oxidized glutathione will accept
hydrogen from reduced TPN but not DPN: the reverse re-
action has not yet been demonstrated [6]. The activity of
many enzymes is inhibited by substances which react with
or oxidize thiol groups, and it is therefore possible that GSH
is part of the mechanism whereby these enzymes are main-
tained in or brought into an active state in vivo. Glutathione
takes an active part [34] in the conversion of methylglyoxal
to lactic acid, a reaction catalysed by the enzyme system
glyoxalase, found for example in Esch. colt and Sac. cere-
visiae. Racker and Krimsky [35] have shown that GSH is
tightly bound to the enzyme triosephosphate dehydrogenase,
and they suggest that a thiol ester of 3-phosphoglyceric acid
is an essential intermediate stage in the formation of 1:3-
diphosphoglyceric acid (cf. the role of Co. A in the pyruvic
oxidase system). Certain reactions in which GSH partici-
pates as a substrate rather than as a co-factor have recently
aroused great interest because of their potential significance
in the synthesis of peptides and proteins in vivo. Working
with cell-free preparations of sheep kidney, Hanes, Hird and
Isherwood [16] have demonstrated that the y-glutamyl
group of GSH and other y-glutamyl peptides (but not
glutamine) can be transferred to peptides or to amino-acids,

lOO NITROGEN METABOLISM ■

i.e. the carboxyl moiety of a peptide bond can be transferred
to a suitable amino acceptor. Thus incubation of GSH with
phenylalanine or tyrosine resulted in the formation of y-
glutamylphenylalanine and y-glutamyltyrosine respectively.
Evidence that new peptides had been synthesized was first
obtained by paper chromatography, and some of these com-
pounds have now been isolated and characterized. The term
transpeptidation has been applied to such transfer reactions
and similar results were later obtained with Pr. vulgaris [37],
Hanes et al. noted that prolonged incubation tended to pro-
duce complete hydrolysis of all the peptides in the experi-
mental system: it is therefore possible that these transpep-
tidation reactions are catalysed by the intracellular proteases
and indeed various proteases, like several other hydrol)rtic
enzymes, are known to be capable of performing transfer
reactions (pp. 104-5).

Utilization of peptides by micro-organisms

Information concerning the utilization of peptides comes
mainly from the response of nutritionally exacting organisms
to peptides containing an amino-acid essential for growth.
The majority of these studies have been performed with
non-proteolytic species and provide evidence that hydro-
lysis by extracellular proteases is not an obligatory step in
the utilization of simple peptides. Using four mutants of
Esch. coli, exacting towards phenylalanine, tyrosine, proline
and leucine respectively, Fruton and Simmonds compared
growth in the presence of simple dipeptides containing the
required amino-acid with that in the presence of the free
acid [12]. Peptides of phenylalanine or tyrosine were as
eifective as equimolecular amounts of the uncombined acids
and there was little or no difference in the growth curves. It
was concluded that prior to utilization, these peptides were
hydrolysed by intracellular peptidases at a rate which did
not limit growth. Lactobacillus arabinosus and Ln. mesen-
teroides likewise utilize dipeptides of glutamine as readily
as free glutamine or glutamic acid [45]. The leucineless
Esch. coli mutant grew at the expense of peptides containing
leucine, but although the rate of grov^^h in the log phase and

PEPTIDES AND PROTEINS lOI

the total amount of growth were equivalent to those on un-
combined leucine, the length of the lag period was increased
in proportion to the concentration of the peptide in the
medium. This effect was not due to time being required for
the formation of an adaptive enzyme. In experiments with
Lactobacillus delbruckii, Lb. casei and Strep, faecalis, other
workers have observed that the utilization of di- and tri-
peptides serving as sources of valine or leucine was affected
by the position of the amino-acid in the peptide, the nature
of adjacent amino-acids, and in some instances the composi-
tion of the medium [i, 23]. Unlike the examples described
so far, the total amount of growth of the proline requiring
Esch. coll mutant was greater when the amino-acid was sup-
plied in the form of a dipeptide, yet the rate of growth was
not affected. A possible explanation is that enzymes which
decompose amino-acids are unable to attack those bound in
peptides, consequently if as a result of peptidase activity an
amino-acid gradually becomes available over a period of
time, a greater proportion will be used in anabolic systems
than if it is all initially present in the free state. Two other
observations support such a conclusion. With Strep, faecalis ,
an organism with an active arginine dihydrolase system
(p. 26), the same amount of growth was produced by appre-
ciably less arginine when it was supplied in the form of small
peptides. Similarly if the organism developed an active tyro-
sine decarboxylase, dipeptides of tyrosine evoked greater
growth than an equivalent amount of free tyrosine [20]. In
a medium containing D-alanine in place of pyridoxin. Lb.
casei becomes exacting towards dipeptides containing L-
alanine, because the D-isomer inhibits the normal utilization
of uncombined L-alanine, another essential nutrilite. High
concentrations of glycine prevent the utilization of D-alanine,
but in the presence of pyridoxin, neither glycine nor D-ala-
nine is inhibitory and there is no requirement for L-alanyl
peptides [21].

Unidentified growth factors believed to be peptides

The growth of several nutritionally exacting bacteria
appears to be dependent on, or is stimulated by, unidentified

102 NITROGEN METABOLISM

substances which are beHeved to be peptides. Before at-
tempting to decide whether such requirements are absolute
it should be noted that in many instances the nature of the
response is determined by the composition of the medium
and the period for which the cultures are incubated [22].
Strepogenin is the name given to acid-labile material pre-
sent in enzymic digests of proteins and required for the
growth of certain streptococci and Lh. casei in synthetic
media. The strepogenin activity of protein digests cannot
be explained solely in terms of their content of glutamine
or asparagine since neither of these substances replaced the
digest factor for a strain of Strep, faecalis [48] and the res-
ponse of Lb. casei to glutamine was different from that to
strepogenin [47]. Moreover, the activity of both glutamine
and GSH, unlike strepogenin, was destroyed by autoclaving.
From their experiments with digests of crystalline insulin,
Sprince and Woolley concluded that glutamic acid and
glycine are two components of strepogenin, and of a large
number of synthetic peptides, only tripeptides exhibited any
activity, serylglycylglutamic acid being the most effective,
though none was as active as the protein digest factor(s).
Strepogenin was antagonized by peptides containing as-
partic acid and also by lycomarasmin, a peptide secreted
by Fusarium ly coper sici and responsible for the wilting of
tomato plants. Lycomarasmin is composed of asparagine,
glycine and a-hydroxyalanine, with the two latter sharing
a common nitrogen atom, but the detailed structure is not
known. From these various experiments, it seems likely that
strepogenin contains a y-glutamyl residue and perhaps func-
tions as a stable source of such groups for the synthesis of
various co-factors. It is worthy of note that organisms res-
ponding to strepogenin also readily decompose glutamine,
and this may be another example of an essential nutrient
being utilized more efficiently when it is supplied in the
form of a peptide. The connection between strepogenin and
the dicarboxylic amino-acids and their amides is, however,
far from clear [cf. 42], and it is not yet possible to account
for all the experimental results. Gravis and intermedius
strains of Corynehacterium diphtheriae also require peptide

PEPTIDES AND PROTEINS I03

growth factors of unknown structure, and some similarities
with strepogenin are indicated [5].

Synthesis of peptides and proteins

Owing to the paucity of available information the syn-
thesis of peptides and proteins can only be discussed in
general terms. Several suggestions have been made as to the
mode of formation of peptide bonds and the three most
likely mechanisms are those concerned with (i) the direct
utilization of metabolic energy, (ii) transfer reactions and
(iii) the reversal of proteolysis.

Calculations based on the synthesis of dipeptides in water
and on the hydrolysis of dipeptides to ionic products have
shown that the free energy associated with the peptide bond
is in the range 420 to 3,000 calories [8, 25]. Though the pre-
cise amount may be the subject of dispute, it is clear that
the de novo synthesis of a peptide bond is endergonic, and
it is reasonable to suggest that in biological systems ATP
functions as a source of energy for the synthesis of peptide
bonds. Glutathione, glutamine, acetylsulphanilamide and
hippuric acid are all simple compounds containing peptide
or peptidic bonds and the synthesis of each of these sub-
stances was first observed in actively respiring preparations
of animal tissues. Any condition inhibiting respiration and
in consequence the production of energy, also inhibited
synthesis. The recognition of ATP as a biological carrier of
energy enabled the experimental systems to be greatly sim-
plified and studies with non-respiring cell-free preparations
revealed that synthesis was dependent on the presence of
ATP. It was observed that a new co-factor, termed Coen-
zyme A, played an important role in the synthesis of acetyl-
sulphanilamide. Coenzyme A is now known to contain
/5-mercaptoethylamine and to function in vivo as a carrier
of acetyl and other acyl groups by virtue of its ability to form
thiol esters. Studies with cell-free systems have shown that
in addition to being synthesized from acetate in the presence
of ATP, acetyl-Co.A can also be formed directly from an
acetyl donor of suitable potential without the intervention
of ATP, e.g. from pyruvate by the pyruvic oxidase system

104 NITROGEN METABOLISM

1

[see 32]. Dried cell preparations of Clostridium kluyveri cata-
lyse the acetylatioh of amino-acids by acetylphosphate, a
reaction which, although analogous to the synthesis of
acetylsulphanilamide, only occurs in the presence of o-i m.
cyanide. Acetylated amino-acids contain a peptidic bond,
and it is feasible that peptides can be formed by transfer
reactions in which the acetyl group is replaced by an amino-
acid [40]. Cell-free extracts of Staph, aureus [9] and a number
of other bacteria [13] and Sac. cerevisiae catalyse the syn-
thesis of glutamine from glutamic acid and ammonia in the
presence of ATP and Mg"^"^ or Mn"^"^. For each mole of
amide synthesized, one mole of inorganic orthophosphate
is liberated, and if ammonia is replaced by NHgOH the pro-
duct is y-glutamylhydroxamic acid. By analogy with the role
of Co. A as a carrier of acetyl groups in the synthesis of
acetylsulphanilamide, it is tempting to suggest that glut-
amine synthesis involves the formation of the corresponding
y-glutamyl compound, but all attempts to obtain supporting
evidence have failed. Though both glutamine and gluta-
thione contain the y-glutamyl radical, there is no proof that
their synthesis involves a common enzyme system or that
the former participates in the synthesis of the latter. When
incubated with ATP, K"^, Mg"^"*", phosphate and hexose
diphosphate, cell-free extracts oiEsch. co/z synthesized GSH
from glutamic acid, glycine and cysteine [37]. Whilst earlier
and similar experiments with preparations of rat liver
showed that the enzymes which synthesized GSH are dis-
tinct from those catalysing hydrolysis, the individual steps
in the biosynthesis are not yet known; the first one may be
the formation of y-glutamylcysteine [38].

In addition to synthesis at the direct expense of metabolic
energy, new peptide bonds may be formed by transfer
(transpeptidation) reactions of the type:

XCO.NH.R+NH2R1 ^ X.CO.NH.Ri+NHoR

where XCOOH, R.NHg and R^.NHa represent amino-acids
or peptides. Such reactions do not result in an overall in-
crease in the number of peptide bonds, and since the type

PEPTIDES AND PROTEINS IO5

and number of bonds in the products is the same as in the
reactants, they proceed with Httle overall change in free
energy and are therefore independent of the availability of
metabolic energy. Several typical proteol}1;ic enzymes are
known to catalyse transpeptidation reactions, and further-
more, to lengthen a peptide chain by the direct coupling of
peptides. Thus, with chymotrypsin [ii]:

benzoyl-L-tyrosinamide+glycinamide ^

benzoyl-L-tyrosylglycinamide+NHs
benzoyl-L-tyrosine+glycinamide ?^

benzoy 1-L-ty rosylgly cinamide + H 2O

It is therefore possible, as suggested many years ago, that
the action of the proteases is reversible and that in the
appropriate conditions they catalyse the synthesis and not
the hydrol^'sis of peptides and proteins. Transpeptidation
reactions involving GSH have already been described (p. 99).
Because cysteinylglycine is readily hydrolysed by cellu-
lar enzymes and yet is relatively stable when combined, as
in glutathione, Hanes and his colleagues proposed that the
attachment of a y-glutamyl radical to a peptide confers re-
sistance to hydrolysis by intracellular proteases, and in con-
sequence, synthesis is favoured and the peptide chain can
be gradually lengthened by successive transfer reactions.
Several bacteria catalyse exchange reactions between the
amide group of asparagine or glutamine and hydroxylamine
or isotopically labelled NHt: with NHgOH, such reactions
lead to the formation of aspartyl- and glutamyl-hydroxamic
acid respectively [45]. Whether the amide group can likewise
be replaced by an amino-acid or peptide is not known. Pro-
tein synthesis may therefore be visualized as a stepwise
process beginning with glutathione, and possibly glutamine,
as a source of peptide bonds synthesized at the expense of
energy derived from ATP, and by means of transpeptidation
reactions the amino-acid components of such bonds are
subsequently altered so as to form peptides from which
specific proteins are synthesized by further transfer and
coupling reactions [11].

I06 NITROGEN METABOLISM

The mechanisms operative in the formation of peptide
bonds are only one aspect of protein synthesis; it is also
necessary to consider (i) the means whereby the correct
sequence of amino-acids is attained in a peptide chain, (ii)
if the protein molecule comprises more than one peptide
chain, how such chains are linked together and (iii) the
spatial arrangement of the amino-acids and the peptide
chains. Major advances in solving these problems await the
determination of the structure of specific proteins (cf.
Sanger's recent elucidation of the amino-acid sequence in
the peptide chains of insulin). Investigations of protein syn-
thesis in animal tissues have been mostly confined to an
examination of the conditions in which isotopically labelled
amino-acids are incorporated into material precipitated by
trichloracetic acid, i.e. presumably bound in proteins or
polypeptides. Such experiments have proved little except
that incorporation is associated with the utilization of meta-
bolic energy, and great care is required in making deduc-
tions from the observed results [49]. A more direct approach
is provided by studies of the synthesis of a specific protein,
e.g. an enzyme whose activity can be estimated and used as
an index of concentration. Evidence has gradually accumu-
lated to the effect that at least in certain cases the adaptive
formation of enzymes is the outcome of de novo protein syn-
thesis rather than the mere subtle modification of existing
proteins, i.e. enzymes or 'enzyme precursors' [30]. If this is
true, then the formation of adaptive enzymes would appear
to offer a most promising field for studying protein synthesis
in micro-organisms. Concentrations of 2:4-dinitrophenol
and azide which, although not affecting respiration and the
fermentation of carbohydrates, inhibit the uptake of inor-
ganic phosphate and in consequence the synthesis of energy-
rich phosphate bonds, also inhibit adaptive enzyme forma-
tion and the incorporation of isotopically labelled amino-
acids into peptides and proteins.

Certain observations indicate that the assembling of the
constituent amino-acids is a preliminary step in the synthesis
of a protein. For example, the synthesis of the adaptive
enzyme nitratase in washed cells of Esch. coli [33] and of

PEPTIDES AND PROTEINS ' IO7

amylase by pigeon pancrease is enhanced by the addition of
amino-acids, and the greater the number of amino-acids, the
greater their effect. Gale has recently studied the effect of
other amino-acids, and of purines and pyrimidines on the
absorption, accumulation and further metabolism of glu-
tamic acid by Staph, aureus, and obtained evidence that an
increase in cellular combined glutamic acid is indicative of
the synthesis of new protein [14]. Protein synthesis only
occurred when the cells were suspended in a medium which
contained, in addition to glutamic acid and glucose, all the
amino-acids to which Staph, aureus is exacting.

Studies of actively dividing embryonic cells and cells
engaged in rapid protein synthesis led Caspersson and inde-
pendently Brachet to propose that protein synthesis is pre-
ceded by the synthesis of pentose nucleic acids and that
these substances then participate in and control the synthesis
of proteins. By using the ultraviolet light microscope tech-
nique (p. 130), Malmgren and Heden measured the nucleic
acid content of cells at various stages during the growth of
cultures of Esch. coli and Bacillus cereus. Their results indi-
cated that the lag phase was a period of intense nucleic acid
synthesis and in consequence the cellular concentration of
nucleic acid reached a maximum during the early part of the
lag phase: thereafter it gradually declined and became mini-
mal during the stationary phase. Malmgren and Heden con-
cluded that Caspersson's and Brachet's hypothesis also
applied to bacteria and that a culture only passed out of
the lag phase when a critical intracellular concentration of
nucleic acid has been attained [28]. Other workers using
Staph, aureus have provided further evidence in support of
this conclusion [14, 29]. The rate of protein synthesis by
washed cells of Staph, aureus can be directly correlated with
the nucleic acid content of the cells at the time of harvesting
[14]. During the growth of bacterial cultures it is the pentose
nucleic acid content of the cell which alters: the desoxy-
pentose nucleic acid content remains approximately con-
stant [29]. It is interesting to note that after irradiation with
ultraviolet light, bacteria are unable to develop the usual
adaptive increase in activity when they are incubated in the

I08 NITROGEN METABOLISM

presence of the specific substrate [44], and that the action
spectrum for light of different wavelengths resembles the
absorption spectra of the nucleic acids [43].

Such experiments have naturally focused attention on the
possible role of the nucleic acids in protein synthesis and so
far two theories have been proposed: one suggests that the
bond energy of the nucleic acid phosphate groups is used
for the synthesis of peptide bonds, whilst the other regards
the nucleic acids as being the fundamental components of
the organized systems controlling the sequence in which
amino-acids are joined together. Proteases appear to possess
well-defined specificity with regard to the peptide bonds
they attack (p. 113), and if they can in fact function syntheti-
cally the same specificity is to be expected in the reverse
reactions. If such enzymes can be organized so that they act
in a predetermined sequence, a mechanism can be envisaged
which possesses the ability to synthesize a peptide chain with
the required serial arrangement of amino-acid residues.
Since nucleic acids differ in their composition and structure
and readily form complexes with proteins, it is possible that
different nucleic acids combine specifically with different
proteins. Hence an organized system of nucleic acids may
provide a framework on to which enzymes are adsorbed in
a particular order, with the result that they then direct the
synthesis of a specific substance [17]. It is also feasible that
the nucleic acids are structures on to which amino-acids
rather than enzymes are adsorbed, and that differences in
nucleic acid structure give rise to different sequences of
amino-acids [3]. If there is any truth in such speculations,
it is to be expected that disruption of nucleic acid meta-
bolism will immediately result in the cessation of protein
synthesis. It could therefore be argued that the effects of
ultraviolet light described above are due to disruption or
degradation of the organized nucleic acid systems directing
adaptive enzyme synthesis. Bacteriophage are composed of
nucleoprotein and they can infect and reproduce in irradi-
ated cells of Esch. coli, although such cells are unable to
form the adaptive enzyme ^^-galatosidase [19], and this may
be construed to mean that irradiated cells can still synthesize

PEPTIDES AND PROTEINS I09

proteins provided the appropriate nucleic acid framework is
made available.

By using materials enriched with radio-active phosphorus
(P^2), several workers have measured the turnover rate of
nucleic acid phosphate groups and the effect thereon of
variations in the rate of protein synthesis. All the results
reported so far are based on isotopic analyses of impure
preparations of nucleic acid and must therefore be treated
with caution since Davidson and his colleagues have shown
that unequivocal results are obtained only if these substances
are rigorously freed from contaminating materials before de-
termining their P^^ content [7]. Spiegelman and Kamen [39]
have found that the fermentation of glucose by washed sus-
pensions of yeast previously grown in the presence of in-
organic phosphate enriched with P^^ did not cause any
decrease in the concentration of P^^ in the nucleic acid
fraction of the cells. However, if NHt was added to the
system, protein synthesis and budding occurred and the P^^
content of the nucleic acids decreased. A similar decrease
took place during the adaptive formation of maltase. The
proposal was therefore advanced that the energy in the phos-
phate bonds of the nucleic acid could be utilized in transfer
reactions for the synthesis of peptide bonds.

From this brief survey it will be apparent that whilst there
is some experimental evidence that nucleic acids mediate
protein synthesis, our present conceptions of the cellular
organization controlling and bringing about such syntheses
are purely speculative (see [51] for a critical analysis of pos-
sible mechanisms for the synthesis of proteins).

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594

8. Dobry, A., Fruton, J. S. and Sturtevant, J. M. (1952), J. hiol.

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9. Elliott, W. H. and Gale, E. F. (1948), Nature, 161, 129

10. Fildes, P. and Gladstone, G. P. (1939), Brit. jf. exp. Path., 20,

334

11. Fruton, J. S., Johnston, R. B. and Fried, M. (195 1), jf. bioL

Chem., 190, 39

12. and Simmonds, S. (1949), Cold Spr. Harb. Symp., 14, 55

13. Fry, B. A. (1954). In the press

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Biochem. J., 51, 25

17. Hokin, L. E. (1952), Biochim. Biophys. Acta, 8, 225

18. Hughes, D. E. (1949), Biochem. J., 45, 325

19. Jacob, F., Torriani, A. N., and Monod, J. (195 1), C.R. Acad.

Sci., Paris, 233, 1230

20. Kihara, H., Klatt, O. A. and Snell, E. E. (1952), J. biol. Chem.,

197, 801

21. and Snell, E. C. (1952), jf. biol. Chem., 197, 791

22. Kodicek, E. and Mistry, S. P. (1952), Biochem.J., 51, 108

23. Krehl, W. A. and Fruton, J. S. (1948), J. biol Chem., 173, 479

24. Lerner, E. M. and Mueller, J. H. (1949), J- biol. Chem., 181, 43

25. Lipmann, F. (1949), Fed. Proc, 8, 597

26. Mcllwain, H. (1948), jf. gen. Microbiol., 2, 186

27. Roper, J. A. and Hughes, D. E. (1948), Biochem. J., 42,

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32. Ochoa, S. and Stern, J. R. (1952), A?in. Rev. Biochem., 21, 547

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Abstracts, p. 93

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PEPTIDES AND PROTEINS III

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Symp., ed. Fildes, P. and van Heyningen, W. E., Cambridge
University Press, G.B.

CHAPTER VIII

PROTEOLYTIC ENZYMES

Putrefaction of the remains of dead animals and plants in
natural environments is due in part to autolysis, i.e. to dis-
integration of cellular components by the organism's own
enzymes [27], and in part to the activities of the mixed
population of micro-organisms which rapidly becomes
established in such conditions. Because of their insolubility
or molecular size, many cellular materials must be degraded
to simpler substances before they can be utilized by micro-
organisms as sources of carbon, nitrogen and energy. De-
gradation may be accomplished either by enzymes present
in the cell wall of the micro-organism, in which case the
organism must itself come into physical contact with the
substrate (e.g. the digestion of cellulose by species of Cyto-
phaga), or by extracellular enzymes produced by the micro-
organism yet acting independently of the parent cell. One
group of extracellular enzymes especially important in
putrefaction is the proteases, enzymes which hydrolyse
peptide bonds and thus make available the amino-acid con-
stituents of proteins.

For several years bacteriologists have used physical mani-
festations of proteolytic activity in the classification and
identification of micro-organisms, e.g. the liquefaction of
gelatin or the clotting and digestion of milk. Moreover, the
unwelcome effects of some pathogenic bacteria are now
known to be due to extracellular toxins which possess en-
zymic activity against certain proteins in the susceptible
host. Mainly because of the influence of industry and medi-
cine, the proteolytic activity of bacteria has been studied
almost exclusively in terms of natural substrates such as
casein, collagen (or gelatin) and fibrin, and more from the
viewpoint of the bacteriologist rather than the biochemist.
On the other hand, there have been considerable advances
in knowledge with regard to animal proteases, several of

112

PROTEOLYTIC ENZYMES II3

which have been crystallized, and a summary of the more
important results of this work provides a background against
which the limited information concerning microbial pro-
teases can be considered.

Hydrolysis of proteins and peptides by animal enzymes [36]

Until 1935 the animal proteases were classified according
to molecular size of substrate and pH for optimum activity,
i.e. the emphasis was on physical properties. Proteins were
regarded as being the substrates of the proteinases (pepsin,
chymotrypsin and trypsin), whilst peptides, substances with
a relatively small number of peptide bonds, were the sub-
strates of the peptidases (aminopolypeptidases, carboxypoly-
peptidases and dipeptidases). The introduction by Berg-
mann of a new and relatively simple method for the chemical
synthesis of small peptides of known composition stimulated
a detailed inquiry into the specificity of enzymes capable of
hydrolysing peptide bonds. By using these synthetic sub-
strates it was shown that the main factors affecting whether
a proteolytic enzyme hydrolysed a peptide bond were,
firstly, the nature of the amino-acids linked together by the
bond, and secondly, the absence or presence of free amino
or carboxyl groups in the vicinity of susceptible bonds.
Bergmann proposed that the proteases be grouped into the
endopeptidases and the exopeptidases according to whether
they hydrolysed peptide bonds remote from or near to the
ends of peptide chains in natural substrates. Endopeptidases
were typified by pepsin, trypsin and chymotrypsin: the
activity of pepsin and trypsin is inhibited by free amino
groups near to susceptible bonds whilst chymotrypsin, and
possibly trypsin as well, is inhibited by neighbouring car-
boxyl groups. The exopeptidases comprise the dipeptidases
and the amino- and carboxy-polypeptidases. The latter two
groups of enzymes attack peptide bonds adjacent to, and in
some cases penultimate to, terminal amino-acid residues
with free amino groups and free carboxyl groups respec-
tively. Whereas both proteins and peptides can serve as sub-
strates for the endopeptidases, the exo_peptidases only
attack peptides. The endopeptidases, unlike some of the

114 NITROGEN METABOLISM

exopeptidases, cannot hydrolyse bonds involving D-amino-
acids. Bergmann and Fruton's investigations appeared to show
that the specificity of the endopeptidases with regard to the
amino-acid composition of the bonds they attacked was rela-
tively high, and this was particularly so in the case of pepsin.
But as more peptides have been synthesized and tested [see
2], it has become clear that these enzymes are not so specific
as the results of the earlier work might suggest. Though few
of them have yet been purified, it is evident that there are
a number of dipeptidases, amino- and carboxy-polypepti-
dases and it is possible that each one has different specificity
requirements.

In addition to the extracellular enzymes, there are a num-
ber of intracellular animal proteases known as kathepsins, of
which four types are known, analogous to pepsin, trypsin,
amino-polypeptidases and carboxy-polypeptidases respec-
tively as regards their specificity. Unlike the extracellular
enzymes, the kathepsins are only active in the presence of
an activator such as HgS, cyanide, cysteine or glutathione,
and in this respect they are very similar to some of the
intracellular proteases of micro-organisms.

Great interest has been taken in the mammalian extra-
cellular proteases known to be secreted in the form of an
inactive precursor, a zymogen, the crystalline form of which
is different from the corresponding active enzyme. Pepsino-
gen is converted to pepsin by treatment with acid or with
pepsin itself, whilst trypsinogen is activated by enterokinase
or by trypsin. Activation of the precursor may involve the
removal of a small peptide (as with pepsinogen) or the open-
ing of a small number of peptide bonds (chymotrypsinogen
and trypsinogen).

There is at present conflicting evidence as to the manner
in which proteins and peptides are hydrolysed by the endo-
peptidases to smaller units. The enzyme may attack all the
susceptible peptide links in any one peptide chain simul-
taneously and thus release the component amino-acids and
peptides concurrently (the 'all or none' hypothesis). Alterna-
tively, it may attack the peptides in a random manner, hydro-
lysing only one bond at a time in any one peptide, until the

PROTEOLYTIC ENZYMES II5

system eventually contains no more susceptible bonds.
Tiselius, on the basis of an electrophoretic analysis of the
reaction mixture obtained by the treatment of egg-white
with pepsin, supported the former concept [39]. However,
chromatographic analysis has revealed that though the sys-
tem ultimately contained mostly tripeptides, a few dipep-
tides, no free amino-acids and still some undigested pro-
tein, in the initial stages of the reaction, deca- and higher
peptides were present [31].

Proteases of micro-organisms

Only a small number of the proteases of micro-organisms
have been purified and only one has been crystallized [14].
Apart from showing that they produce certain effects, e.g.
the liquefaction of collagen and gelatin, there have been few
attempts to express activity in terms of the hydrolysis of
peptide bonds, and virtually none concerned with their
specificity with respect to the bonds attacked [32]. Most of
the experiments with synthetic substrates have been con-
fined to the peptidases and no bacterial proteinase has been
examined in such detail as mammalian pepsin. In addition
to intracellular proteases, some micro-organisms also pos-
sess extracellular proteolytic enzymes and these enable pro-
teins in the environment to be degraded at least to small
peptides, if not to amino-acids, and the products are then
absorbed and may be further degraded by intracellular
enzymes.

The biological activity of extracellular substances pro-
duced by micro-organisms can be detected by incorporating
the appropriate substrate in the culture medium or, more
usually, by testing the activity of the culture filtrate. The
test may be applied directly to the latter, or to a concentrate
of the active principle prepared from the culture filtrate by
evaporation in vacuo^ by precipitation with ammonium sul-
phate or ethanol, by freeze drying, or by an adsorption
technique. Enzymic activity of the experimental material is
detected by incubating with the appropriate substrate, toxic
activity by injecting the material into animals or by pre-
cipitation reactions with antisera, and antibiotic activity by

Il6 NITROGEN METABOLISM

incorporating the material into culture media. The occur-
rence of enzymic activity in culture filtrates must not be
taken as unequivocal evidence that the enzyme is extra-
cellular, particularly if high activity is dependent on pro-
longed incubation of the cultures: in such circumstances it
is natural to suspect that the organisms may have undergone
partial autolysis and thus released intracellular enzymes into
the medium. If a culture filtrate contains more than one
protease, they may be separated from one another by the
usual procedures employed in the purification of enzymes.
Very active extracellular proteases are produced by species
of Proteus, Clostridium, Bacillus, Pseudomonas and Micro-
coccus', less active enzymes are formed by the streptococci
and staphylococci whilst the lactobacilli and Enterobacteri-
aceae (with the exception of Proteus spp.) apparently pro-
duce none.

Collagenase and gelatinase activity

The ability of certain bacteria to liquefy collagen and the
material derived from it, gelatin, was soon discovered fol-
lowing the introduction of gelatin as a means of solidifying
culture media [12]. One of the methods employed in the
quantitative determination of gelatinase activity makes use
of an Ostwald viscometer [13], the method being based on
the assumption that changes in the viscosity of a solution of
gelatin are an index of proteolytic activity. Such a procedure
is indirect, and the observed changes in viscosity may not
necessarily be due to the hydrolysis of peptide bonds. It is
therefore desirable that viscometric methods should be com-
pared with those that are based more directly on the results
of proteolytic activity, and in particular on the expected
appearance of free amino and carboxyl groups in the system.
Suitable methods of this type employ the van Slyke appar-
atus for the determination of amino groups and the Sorensen
titration procedure for carboxyl groups. Indeed, it is evident
from the work of Gorini and Fromageot that changes in
viscosity may bear no relation to the appearance of free
amino groups (Fig. 8.1), but such a correlation has not often
been attempted.

PROTEOLYTIC ENZYMES II7

The most detailed studies of the proteolytic enzymes in
bacterial culture filtrates are those of Maschmann [29], who
isolated from the culture filtrates of the invasive Clostridia
{CI. histolyticum, CI. welchii, CI. septicum) an enzyme that
attacked gelatin and collagen but had no action on casein,
peptone or egg albumin. Even filtrates from young cultures

600-t

h^SOO-

5400-

0 300-

^lOO-

'l8hr.)

K

(18 hr.)

O

(20 min)

X

-24-0

-160 >.

«n
O

■120 o>
>
Ui

>
hQO P

/

cor

(Ihr.)

-40

I 1 \

^ 0-5 I'D , 1-5 20

ENZYME CONC. (ml. soln.)

FIG. 8.1. — Hydrolysis of gelatin by extracellular proteinase of
Micrococcus lysodeikticus as a function of enzyme concentra-
tion. Activity measured by appearance of amino nitrogen
(solid line) and by changes in viscosity (broken line). Reaction
times shown in parentheses. (From Gorini, L. and Fromageot,
C. (1950), Biochim. Biophys. Acta, 5, 524; Elsevier Publishing
Co. Inc., New York and Amsterdam)

contained this specific gelatinase, and it was therefore re-
garded as being a truly extracellular enzyme. The invasive
properties of certain Clostridia can be ascribed to their ability
to secrete this enzyme since Maschmann could not detect
any specific gelatinase in filtrates from the non-invasive
Clostridia {CI. botidinum, CI. tetani), and later workers
showed that the culture filtrates of the invasive Clostridia
digested the collagen supporting material of muscle whereas
an enzyme such as trypsin liquefied the muscle fibrils and

Il8 NITROGEN METABOLISM

left the connective tissue intact [34, 15]. The collagenase of
CI. welchii type A, also known as the kappa (k) toxin, has
been purified considerably [11]. Although on a weight basis
this material was less toxic than the lecithinase (a-toxin) of
CI. welchii, it was ten to fifty times more toxic than the
endotoxins of Gram-negative bacteria. A curious feature of
the purified collagenase is that after exposure to mild alkali
or to heat, it still attacked gelatin but not collagen [9]. This
treatment may have altered the structure of the enzyme such
that it can combine only with gelatin, and this may also be
the reason why there is a change in the optimum pH for
gelatinase activity. Another possible, though perhaps un-
likely, explanation is that exposure to heat or alkali de-
natured the collagenase and at the same time activated a
specific gelatinase precursor [9]. Some confusion exists in
the naming of the enzymes which attack gelatin and collagen
and it is sometimes assumed that the terms gelatinase and
collagenase are synonymous. However, Evans and Wardlaw
have evidence that though the formation of collagenase by
various species of bacilli is always accompanied by gelatinase
activity, some organisms, e.g. B. subtilis, produce a gelatinase
and yet have no apparent action on collagen [16].

Other proteinases of the Clostridia

Apart from the specific gelatinase, Maschmann found
three other types of proteolytic enzymes in various bacterial
culture filtrates (Table 8.1). One type was active against pro-
teins and peptones, and like the specific gelatinase, was found
in young as well as old cultures and activity was unaffected
by the presence of Og: gelatin was also digested, but usually
not as rapidly as casein. The two other types of proteases,
one active against proteins, the other against peptides, ap-
peared in the medium after the cultures had stopped grow-
ing, and it is therefore possible that at least in some instances
they were intracellular enzymes released by autolysis. Their
activity was depressed by O2 and was only maximal in the
presence of reducing substances (HgS, cysteine or gluta-
thione) whilst in addition, the peptidases also required di-
valent cations (Mg+"^, Fe++ or Mn++). The Og-labile

PROTEOLYTIC ENZYMES

119

proteinases were found in cultures of CI. welchii, CI. histo-
lyticum, CI. botulinum and CI. septicum; peptidases were
present in cultures of all the organisms named in Table 8.1.
There may be strain differences with regard to the enzymes
formed by a particular species. For example, the proteinase
of CI. histolyticum isolated by Maschmann w^as distinguished
from that isolated by later workers in not being activated by
cysteine. Further investigations revealed that some strains
of CI. histolyticum produced both of these proteinases [23].

TABLE 8.1

PROTEOLYTIC ENZYMES OF ORGANISMS STUDIED BY MASCHMANN

Organism

Specific
gelatinase

Proteases, attacking casein

and peptone, inhibited

by normal serum

Enzyme
stable in
oxygen

Enzymes
stable in
oxygen

Enzymes
labile in
oxygen

CI. botulinum

CI. feseri

CI. histolyticum

CI. septicum

CI. sporogenes

CI. tetani

CI. ivelchii

Ps. aeruginosa

Ps. fluorescens

Serratia marcescens

+

+
+

+

+ 1 + 1 + + 1 + + +

+ 1 +++ 1 + 1 1 1

The one activated by Fe"^"^ and thiol compounds attacked
casein and clupein as well as gelatin, and liberated more
carboxyl groups than amino groups, indicating that peptide
bonds involving the amino group of proline had been hydro-
lysed [28]. The lambda (X) toxin of CI. welchii, purified by
Bidwell, is a proteolytic enzyme capable of hydrolysing
gelatin, casein, haemoglobin and hide pow^der, but it has no
effect on native collagen [10]. It is strongly inhibited by
cysteine, and, though there is no evidence that it is activated
by Mg"^"*" or Mn"^"^, by citrate.
9

120 NITROGEN METABOLISM

Maschmann observed that the proteinases, but not the
specific gelatinase, were inhibited by normal (i.e. not neces-
sarily immune) sera. A more recent careful kinetic study of
the proteolytic activity of several bacteria disproved the idea
that this effect wsls due to the same substance that inhibits
trypsin [13]. The trypsin inhibitor is found only in the
albumin fraction of the serum proteins, whilst the labile
antibacterial protease factor is in the globulin fraction:
furthermore, the bacterial enzymes are not inhibited by the
trypsin inhibitors present in soya bean, the pancreas and
ovomucoid. The antibacterial serum factor inhibited all the
bacterial proteases examined, even the specific gelatinases
to a small extent. These investigations of Duthie and Lorenz
also confirmed that the ability to clot milk is restricted to
certain bacteria, and showed that although inhibited by the
globulins of normal sera, the rate of clotting bore no relation-
ship to the protease activity of the culture filtrates.

Factors affecting the formation and activity of extracellular
proteinases
The composition of the growth medium profoundly
affects the degree of proteinase activity that is ultimately
detectable in the culture filtrate. Three factors appear to be
of special significance, (i) inorganic ions, (ii) fermentable
carbohydrate and (iii) the organism's source of nitrogen. A
previous observation [30] that protease production by a
species of Proteus depended on the presence of Ca and
Mg"^"^ was investigated in more detail by Hanes, using the
organisms Bacillus subtilis, B. mesentericus, Pr. vulgaris, Ps.
fiuorescens and Ps. aeruginosa [22]. The addition of Ca^"^
had little effect on grov^h, but markedly increased the pro-
duction of gelatinase, whilst Mg"^"*", although increasing
growth, caused no increase in the gelatinase activity of the
cultures. The recent work of Gorini has served to emphasize
the important role of cations such as Ca"^^ [17]. Optimal
extracellular protease activity of Micrococcus lysodeikticus,
B. megatherium, Proteus, Ps. pyocyanea and B. mesentericus
was dependent on growth at a low temperature (26° C.) in

PROTEOLYTIC ENZYMES 121

well-aerated media in the presence of Ca"^"^. The amount
of glucose added to the medium had to be such that the pH
at the end of growth had not fallen below pH 7. Gorini con-
tends that as well as activating the proteases, Ca"*" "*" also has
a stabilizing influence and that in the absence of Ca"*"^ these
enzymes are inactivated as fast as they appear in the
medium [19]. This view is opposed to that of previous
workers who believed that Ca"^ "*" stimulated the actual pro-
duction of the enzymes. When proteins and polypeptides
were used as a source of nitrogen, the growth of the culture
was dependent on its proteolytic activity, and in such condi-
tions Ca"*"^ was indispensable for growth [i8]. Sodium
fluoride and citrate, substances capable of combining with
Ca"^"^, inhibited these proteases, and although Mg"^"^ pro-
tected them against such inhibitors and from denaturation
by heat, Mg"^"^ could not replace Ca"^"^ as the cationic acti-
vator [17]. There is some evidence that the gelatinase activity
of cultures of B. suhtilis is dependent on the Mn"^"^ content
of the medium [38].

Although several workers have regularly demonstrated
activity in filtrates from cultures grown in media containing
glucose [30], other workers have reported that the presence
of fermentable carbohydrate inhibits the formation of pro-
teolytic enzymes [3]. Such effects are probably to be attri-
buted to the acidic products of carbohydrate catabolism
causing the pH of the medium to fall to a value which does
not favour the formation of proteases. In buffered media, or
in those where the pH does not become acid [cf. 17, 19] the
presence of fermentable carbohydrate has no effect [3].

Several attempts have been made to ascertain whether the
growth of proteolytic bacteria is supported by pure native
proteins, or only by those which have been denatured or
partially degraded. Such bacteria failed to grow when sub-
cultured into a medium containing inorganic salts and a pure
protein as a source of carbon and nitrogen [i]. This might
be explained on the basis that the synthesis and excretion
of an extracellular enzyme involves the utilization of energy,

122 NITROGEN METABOLISM

which in these experiments could only be derived from
amino-acids after they have been made available by enzymic
hydrolysis of the protein. Even if any extracellular enzyme
is carried over in the inoculum it is possible that it is diluted
in the subculture to such an extent that its activity is no
longer significant. If a small amount of peptone, presumably
containing some small peptides or amino-acids, was added
to the medium, certain proteolytic bacteria, e.g. Pr. vulgaris^
grew rapidly and crystalline egg albumin and serum pro-
teins were then readily degraded and utilized [37]. Hence it
appears that the formation of extracellular enzymes is de-
pendent on the medium containing sources of carbon,
nitrogen and energy that can be utilized immediately with-
out having to be first broken down into smaller units by
extracellular enzymes. In any event, proteoses and peptones
are apparently not attacked by members of the Bacteriaceae
or by Staph, aureus or Strep, faecalis [33].

Specific proteinases of the streptococci and staphylococci

The culture filtrates of some Lancefield group A strepto-
cocci contain an Og-labile papain-like enzyme which apart
from hydrolysing fibrin, casein, gelatin and benzoyl-L-
arginamide, also attacked the M antigen, one of the antigens
used in typing group A streptococci [14]. The M antigen is
usually absent in those strains capable of producing this
enzyme, though it may be present if the cultures are grown
at a low temperature (22° C.). After passage through mice,
formation of the enzyme ceased and the organisms became
more virulent and possessed the M antigen. The latter two
effects are not directly related, since the M antigen is also
present in some avirulent strains. Of some clinical impor-
tance is the fibrinolytic activity of haemolytic streptococci
(groups A and C), staphylococci and gas-gangrene Clostridia.
These bacteria produce an enzyme which converts a pre-
cursor (plasminogen) in the globulin fraction of human sera
into an active enzyme (plasmin) which digests the fibrin of
clotted blood. The mode of action of the bacterial enzyme,
named streptokinase in the case of streptococci, is compar-
able with the activation of chymotrypsinogen by trypsin.

PROTEOLYTIC ENZYMES I23

Infection with Strep, haemolyticus soon results in the forma-
tion of an antibody which completely antagonizes strepto-
kinase.

Peptidases of bacteria, aspergilli and yeast [4, 25, 36]

A number of observations have provided evidence for the
occurrence of peptidases in micro-organisms similar to those
in animals and plants, and in general their activity has been
studied using simple substrates, di- and tri-peptides of
glycine, alanine and leucine. Many, but not all, of these
enzymes are activated either by thiol compounds or by di-
valent cations: some require both types of activator (Table
8.2). The peptidases of the Clostridia exhibit poor activity

TABLE 8.2

ACTIVATION OF MICROBIAL PEPTIDASES

The activators listed below are those which have been found to
increase the activity of various preparations of peptidases from the
organisms shown in the table. Which of these substances are the
most effective activators for particular enzyme preparations de-
pends on the peptidase concerned and sometimes on the substrate
being tested.

Organism Activators

Aspergillus parasiticus Zn , cysteine

Bacillus megatherium Zn , Mn , Fe , cysteine

Clostridiu?n histolyticum Mn , Fe , cysteine

Escherichia coli Mg"^"*", Mn"^"^

Leuconostoc mesenteroides TjXi , Mn , Cd , Pb , cysteine

Phytomonas tumifaciens Mg"^"*") Mn , cysteine

Proteus vulgaris Mg""""*", Mn"*"*"

Pseudomonas fluorescens Mg > Mn "^

Saccharomyces cerevisiae Zn"*" , Mn , Fe , Cl~, Br", NO7

except in the presence of cysteine together with a cation such
as Fe"*"^ or Mn"*"*" (concentrations of the order io~^ to
io~^ M.). For the hydrolysis of leucyl peptides, the best acti-
vator was Fe"*"^, for alanyl peptides, Mn"'"'' was better [29].
Maschmann suggested that the active enzyme was formed
by combination of the cation wdth the reduced form of the

124 NITROGEN METABOLISM

'apoenzyme', and that the cation acted as a bridge joining
the enzyme to the substrate. Smith [36] has proposed that
the cation chelates with the substrate, and thus causes a
redistribution of electrons with the result that the suscep-
tible peptide bond becomes unstable and easily broken:

— NH.CH(R).C NH.CH(R')COO—

II
O

e.g. for a
carboxypeptidase,

Enzyme protein

The co-ordination is believed to be between the — CO —
group of the peptide bond and the terminal free carboxyl
group for carboxypeptidases and the — NH — group and
the free amino group for aminopeptidases. Examples are
known in which co-ordination between cations and peptides
is extremely specific, and this may explain why peptidases
attacking different substrates are activated by different
metal ions. The aerobic bacteria Ps. aeruginosa, Ps. fluores-
cens and Serratia marcescens contain a peptidase activated
by Mg"*"^ and comparable in specificity with the leucine
aminopolypeptidase of animal tissues [5, 7]. Dipeptidases,
or enzymes with activity against dipeptides, have been found
in preparations from Mycobacterium tuberculosis [35], Leuco-
nostoc mesenteroides [6], Phytomonas tumifaciens [5], 5. mega-
therium [8], Sac. cerevisiae [20, 21] and Aspergillus para-
siticus [7, 26]. A polypeptidase from Sac. cerevisiae and Asp.
parasiticus was activated by Zn"^"*", and both the di- and
poly-peptidase of yeast also required chloride ions [24]
(Table 8.2). In general, the optimum pH for peptidase
activity is in the range 8-9, but some peptidases of Ln.
mesenteroides, propionibacteria and lactobacilli are most
active at an acid pH (5-5-6-0), and they are not activated
by divalent cations [5, 6].

PROTEOLYTIC ENZYMES I25

REFERENCES

1. Bainbridge, F. A. (19 11), J. Hyg., 11, 341

2. Baker, L. E. (195 1), J. biol. Chem., 193, 809

3. Berman, N. and Rettger, L. F. (1918), J. Bact., 3, 389

4. Berger, J., Johnson, M. J. and Peterson, W. H. (1937). Enzymo-

logia, 4, 3 1

5. (1938), J. Bact., 36, 521

6. (1938), y. biol. Chem., 124, 395

7. (1939), J- biol. Chem., 130, 641

8. ■ (1940), J. biol. Chem., 133, 157, 639

9. Bidwell, E. (1949), Biochem. J., 44, 28

10. • (1950), Biochem. J., 46, 589

11. and van Heyningen, W. E. (1948), Biochetn. J., 42, 140

12. Brunton, T. L. and MacFadyen, A. (1889), Proc. Roy. Soc,

46B, 542

13. Duthie, E. S. and Lorenz, L. (1949), Biochem. jf., 44, 167, 173

14. Elliott, S. D. (1945), J- exp. Med., 81, 573; (1950), 92, 201

15. Evans, D. G. (1947), J. gen. Microbiol., i, 378

16. and Wardlaw, A. C. (1953), jf- gen. Microbiol., 8, 481

17. Gorini, L. (195 1), Biochim. Biophys. Acta, 6, 237

18. and Audrian, L. (195 1), Biochim. Biophys. Acta, 6, 477

19. and Fromageot, C. (1950), Biochim. Biophys. Acta, 5, 524

20. Grassmann, W. (1927), Z. physiol. Chem., 167, 202

21. and Dyckeroff, H. (1928), Z. physiol. Chem., 179, 41

22. Hanes, R. B. (1931), Biochem. J., 25, 1851; (1932), 26, 323;

(1933), 27» 466

23. van Heyningen, W. E. (1940), Bioche?n. jf., 34, 1540

24. Johnson, M. J. (1941), J. biol. Chem., 137, 575

25. and Berger, J. (1942), Advances in Enzymology, 2, 69

26. and Peterson, W. H, (1935), J. biol. Chem., 112, 25

27. Jones, A. S., Stacey, M. and Webb, M. (1949), Biochem.

Biophys. Acta, 3, 383

28. Kocholaty, W. and Krejci, L. E. (1948), Arch. Biochem., 18, i

29. Maschmann, E. (1943), Ergeb. Enzvmforsch., 9, 155

30. Merrill, A. T. and Mansfield-Clark, W. (1928), J. Bact., 15,

267

31. Moring-Claesson, I. (1948), Biochifn. Biophys. Acta, 2, 389

32. Mycek, M. J., Elliott, S. D. and Fruton, J.' S. (1952), J. biol.

Chem., 197, 637

33. Rettger, L. F., Berman, N. and Sturges, W. S. (1916),^ Bact.,

I, 15

34. Robb-Smith, A. H. T. (1945), Lancet, ii, 362

35. Roulet, F. and Zeller, E. A. (1948), Helv. Chim. Acta, 31, 191 5

36. Smith, E. L. in The Enzymes, 1 (ii), Chap. 23

37. Sperry, J. A. and Rettger, L. F. (i9i5),.7. biol. Chem., 20, 445

38. Stockton, J. R. and Wyss, O. (1946), ^ Bact., 52, 227

39. Tiselius, A. and Eriksson-Quensel, I. -B." (1939), Biochem. jf.,

33, 1752

CHAPTER IX

NUCLEOTIDES AND NUCLEIC ACIDS

It requires but a short acquaintance with biochemistry to
reahze that the importance of nucleotides and nucleic acids
is comparable with that of amino-acids and proteins.
A nucleotide consists of a heterocyclic nitrogen compound,
such as nicotinamide, a purine or a pyrimidine, joined to a
sugar to which one or more orthophosphate groups are
attached. The sugar is either a pentose (e.g. ribose) or a
deoxypentose, and the dephosphorylated form of a nucleo-
tide is known as a nucleoside. (The latter is really a N-
glycoside and a nucleotide is therefore a phosphorylated
N-glycoside.) Natural pyrimidines and purines include

/ 4 \

N3 5CH

N3 5C

pyrimidine,

CH2

II
6CH

and

purine,

1 II

CH2 6C

^n/\i^

sCH

cytosine (4-amino-2-ketopyrimidine), uracil (2:4-diketo-
pyrimidine), thymine (5-methyluracil), adenine (4-amino-
purine) and guanine (2-amino-4-ketopurine).^ The corre-
sponding ribose nucleosides are known as cytidine, uridine,
adenosine and guanosine, and the nucleotides as cytidylic,
uridylic, adenylic and guanylic acid respectively. Plants and
animals also contain 5-methylcytosine, but contrary to
earlier reports, this pyrimidine is apparently not present in
micro-organisms [51, 57].

Nucleotides are constituents of nucleic acids and also of
the prosthetic groups and co-factors of many enzyme sys-
tems concerned with transfer reactions (e.g. ATP in energy
transfer; DPN, TPN, FMN and FADN in hydrogen transfer;

^ New system of numbering recommended by the Chemical
Society {J. Chern. Soc, 5064 (1952) )•

126

NUCLEOTIDES I27

Co. A in acyl group transfer). The recently discovered
nucleotide, uridine-diphosphate-glucose, is a co-factor in
the enzymic conversion of galactose- 1 -phosphate to glucose-
I -phosphate [9]. With the exception of FMN, in which the
glycone is ribitol, all these co-factor nucleotides contain
/i-ribose in the furanose form and phosphorylated on €'-5.
In co-factors composed of two nucleotides the internucleo-
tide bond is between the two 5-phosphate groups.

Nucleic acids are composed of large numbers of nucleo-
tides and are consequently compounds of high molecular
weight (of the order 5 x 10^ to i x lo^), but although the indi-
vidual units are relatively simple, the determination of the
detailed structure of a nucleic acid entails the solving of
problems comparable in difficulty with those encountered
in the elucidation of the structure of a protein (cf. p. 106).
Depending on whether the component nucleotides contain
either ribose or 2-deoxyribose, nucleic acids have been
divided into two types, the ribose nucleic acids (RNA) and
the deoxyribose nucleic acids (DNA). In only three instances
has a derivative of ribose or 2-deoxyribose been isolated and
characterized, and in the absence of such evidence some
workers [14] prefer the terms pentose nucleic acid (PNA)
and deoxypentose nucleic acid (DPNA). Adenine, guanine
and cytosine are constituents of all the known nucleic acids,
and in addition a PNA contains uracil, whereas a DPNA
contains thymine. In consequence of their large numbers
of phosphate radicals, nucleic acids are highly acidic and
readily form salt-like compounds with bases. However, at
least in bacteria, the proteins associated with nucleic acids
in nucleoproteins are not necessarily of the basic protamine
or histone type [15].

Structure of nucleotides and nucleic acids

Only in a few instances is there adequate proof of the
structure of the component nucleotides of nucleic acids, but
by analogy it is assumed that they all follow the same general
pattern. The sugar is present as the ^-isomer and in the
furanose form, with a glycosidic linkage between the reduc-
ing group (C'-i) and N-7 of the purines and N-i of the

128 NITROGEN METABOLISM

pyrimidines. Position C'-3 is phosphorylated in the deoxy-
pentose nucleotides, and though it is probably the same in
the pentose nucleotides, position C'-2 is a possible alterna-
tive. Since hydrolysis in certain conditions yielded four types
of nucleotides, apparently in equimolecular proportions, all
nucleic acids were at first thought to be polymers of units
each of which contained the four nucleotides arranged in a
straight chain or in a cyclic tetrad. An alternative suggestion
was that the overall equivalent proportions did not neces-
sarily imply such a regular arrangement but were due simply
to a statistical mean. Such theories required that the ratio
of purine-N to pyrimidine-N should be 2:1, but with the
accumulation of precise quantitative data came the realiza-
tion that few nucleic acids contained the four nucleotides,
or even purines and pyrimidines, in equimolecular propor-
tions. For example, three types of DPNA have been isolated;
in the one found in animals, yeast and most bacteria, adenine
and thymine predominate (AT type), in another, found in
only a few bacteria, guanine and cytosine predominate (GC
type), whereas in the third type isolated from strains of
Esch. colt, the bases are in equimolecular proportions [13].
Although discovered over eighty years ago, detailed in-
vestigations of structure have been confined to only two
nucleic acids, a RNA from yeast and a DRNA from the
thymus, and of these most is known about the former.
Markham and Smith [38] believe that yeast RNA is a mix-
ture of comparatively short, straight chains of nucleotides
in which the internucleotide linkage is between the phos-
phate at C'-3 (or C'-z) of one nucleotide and the hydroxyl
group at C'-5 in the adjacent nucleotide. They have also
presented evidence that some of the chains terminate in
cyclic nucleotides, i.e. nucleotides in which the phosphate
group forms a bridge between C'-2 and €'-3. By using ion
exchange resins, previous workers had shown that each of
the four nucleotides in alkaline hydrolysates of yeast RNA
could be separated into two isomers, the 'a' and *b' nucleo-
tides, which were regarded as being the nucleoside 2'- and
3 '-phosphates. It is now evident that only one of these
isomers occurs naturally, and that the other is formed by

NUCLEOTIDES I29

the hydrolysis of cyclic nucleotides which are either present
initially in the nucleic acid or are produced during the
hydrolysis procedure [38]. The products obtained by the
digestion of RNx\ with ribonuclease are mainly mono- and
di-nucleotides together with some polynucleotide material
which, because it w^ould not dialyse, was at first thought to
be the 'core' of the nucleic acid and to be of high molecular
weight. Markham and Smith have now shown that the 'core'
consists of relatively small polynucleotides whose dialysis
depends on the concentration of salt in the system [38].

Estimation of nucleic acids and their components

The procedures for estimating total nucleic acid are based
on either ultraviolet spectrophotometry or on determinations
of orthophosphate or sugar (pentose and deoxypentose). In
the latter chemical methods the cells are first extracted with
cold trichloracetic acid (TCA) and a fat solvent to remove
acid-soluble compounds and phospholipoids. After the nu-
cleic acids have been released from the nucleoproteins
by heating the extracted cells with 5% TCA at 90° C. for
15 minutes, pentose is estimated colorimetrically by the
orcinol method and deoxypentose by the Dische-diphenyl-
amine method [48]. In the Schmidt and Thannhauser pro-
cedure [47] the extracted cells are treated with N.-KOH at
37° C. to hydrolyse the PNA to free nucleotides, and then
the undegraded DPNA and protein are precipitated by
making the hydrolysate normal to HCl. The DPNA and
PNA content of the original material is calculated from the
organic and inorganic phosphate in the hydrolysate and
the organic phosphate in the DPNA fraction. This method
assumes that the only cellular acid-insoluble phosphorus
compounds are nucleic acids and phosphoproteins — an
assumption now known to be invalid [42]. In both the sugar
and phosphate methods, the results are expressed in terms
of nucleic acid by using conversion factors based on thymus
DNA and yeast RNA as standards.

A characteristic property of purines and pyrimidines is
that they strongly absorb in the ultraviolet region of the
spectrum, with a peak absorption in the region of 260 m^«.

130 NITROGEN METABOLISM

It is therefore natural that spectrophotometric techniques
should have been developed for the quantitative estimation
of the free bases and the various substances in which they
are constituents. The total nucleic acid content of organisms
spread in a film on a slide can be determined by ultraviolet
spectrophotometric microscopy [10], whilst for cell suspen-
sions, there is a technically simpler procedure based on the
use of a standard ultraviolet spectrophotometer [40]. By
making various assumptions, an average value for the ab-
sorption coefficient of a typical nucleic acid can be deduced
and thus the absorption measurements interpreted in terms
of nucleic acid. The originators of these techniques are well
aware that reliable results are only obtained if due account is
taken of a large number of variables and that many of the
basic premises may not be strictly valid [cf. 10, 40]. Never-
theless, even if the results are not entirely accurate in terms
of precise quantitative values, they are extremely useful,
particularly when the data are used in a comparative manner.
The identification and quantitative determination of the
various bases and nucleotides present in a nucleic acid
necessarily involves hydrolysis of the nucleic acid, and
chemical hydrolysis without degradation of one or more of
the nitrogenous bases presents some difficulty. Depending
on the conditions employed, the products are nucleotides,
nucleosides, the free bases or mixtures of these substances.
Thus subjecting a PNA to mildly acidic conditions, e.g.
N.-HCl at 100° C. for i hour, liberates the purines in the
free state, but the pyrimidines still remain in nucleotide
combination from which they are freed by more vigorous
hydrolysis, e.g. formic acid at 175° C. [see 25]. On the other
hand, alkaline hydrolysis of a PNA yields a mixture of the
four nucleotides. The deoxypentose nucleic acids behave
differently on chemical hydrolysis, and degradation to
nucleosides and nucleotides is best achieved by the use of
enzymes. In the past, separation of the end-products ob-
tained by hydrolysis or enzymic digestion of nucleic acid
was accomplished by precipitation as the phosphotungstate
or as the uranium or silver salt, or by simply adjusting the
pH of the system. More recently, ionophoresis on paper [38]

PLATE II. — Separation and identification of the purines and pyrim-
idines in yeast ribonucleic acid (YNA): YNA hydrolysed for
2 hr. in 70 %HC104 at 100° C: solvent system isopropanol
(65% v/v) and 2N-HC1 (final concentration): G, A, C, and
U pure samples of guanine, adenine, cytosine and uracil.
Photograph obtained by placing chromatogram over reflex
document paper and exposing to ultraviolet light. ST. de-
notes starting line

NUCLEOTIDES I3I

and chromatography, both on paper [11] and on ion ex-
change resins [18], have provided more convenient and pre-
cise techniques for the separation, preparation [19] and
identification of nucleotides, nucleosides and the bases.
When a paper chromatogram is exposed to light of wave-
length 260 m^w, the areas occupied by purines, pyrimidines,
nucleosides or nucleotides appear as dark spots on a light
blue fluorescent background. A permanent photographic
record can be obtained by placing the chromatogram over
reflex copying paper [37], and the areas in the chromato-
gram containing compounds absorbing ultraviolet light will
appear as white spots on a dark background in the developed
photograph (Plate II). The appropriate areas of the chro-
matogram are then cut out, the compounds eluted and
estimated spectrophotometrically.

Microbial nucleic acids

Micro-organisms, particularly bacteria, are richer in
nucleic acid than most of the cells of other organisms, and
Belozersky has calculated that 15-30% of the dry weight of
bacteria is nucleic acid and 50-80% is nucleoprotein [4].
Prior to isolating a nucleic acid, soluble nucleotides and
phospholipoids are first removed by successively extracting
the cells with cold TCA and a fat solvent. The residue is
then treated with dilute solutions of an alkali, e.g. 0-2%
NaOH or NagCOg in order to extract the nucleic acids, the
details of the procedure varying according to the nucleic
acid required. The isolation of microbial nucleoproteins,
especially in an undegraded or 'native' state, presents many
difficulties and, apart from low yields, it is doubtful if any
of the present techniques are ideal. The most favoured
method is to extract the cells with neutral solutions of NaCl
at a concentration depending on the type of nucleic acid it
is desired to isolate: even so, the deoxypentose nucleopro-
teins adhere strongly to the cell structure and are only re-
moved with difficulty [4]. When an aqueous solution of a
nucleoprotein is shaken with chloroform, the protein be-
comes denatured and collects at the interface, whilst the
liberated nucleic acid remains in the water phase. The

132 NITROGEN METABOLISM

sodium salt of the nucleic acid can then be precipitated by
the addition of ethanol, or, alternatively, the free nucleic
acid by the addition of acidified ethanol or glacial acetic acid.
The staining procedure introduced by Christian Gram to
reveal the presence of bacteria in animal tissues was subse-
quently developed into an empirical technique for dividing
bacteria into two groups. After the fixed organisms have
been stained with a basic dye (crystal violet or methyl violet)
at pH 7-8, they are treated with a mordant, usually 1 2 in KI,
and then washed with ethanol or acetone. If the stain is
quickly removed, the organism is said to be Gram-negative;
whereas if the dye remains, it is regarded as being Gram-
positive. After the ethanol or acetone treatment, it is now
common practice to counterstain with a red dye, with the
result that in the final preparation Gram-positive organisms
are stained blue whilst Gram-negative organisms are red.
Though the mechanism of this staining reaction is still a
matter of dispute [see 3, 41], retention of the basic dye by
Gram-positive bacteria appears to be due to the presence of
a Mg'^'^-PNA complex in the peripheral layers of the cells.
The evidence for this belief rests on the observation that
after treatment of the dead cells with ribonuclease — an
enzyme depolymerizing PNA (p. 1 34) — or with a detergent
such as bile salts. Gram-positive organisms stain as though
they were Gram-negative. The action of the detergent is to
liberate PNA from the cells and this material has been iso-
lated by Henry and Stacey, who found that in the presence
of Mg"*"^ and a reducing substance, the isolated PNA,
or indeed yeast RNA, would restore Gram-positive stain-
ing properties to the appropriate Gram-negative cytoskele-
tons [21], but not to truly Gram-negative organisms. Though
the ratio of PNArDPNA was reported to be 8:1 in Gram-
positive as opposed to 1-3:1 in Gram-negative bacteria, these
figures are disputed by Mitchell and Moyle who claim that
the ratio is about 4:1 in all bacteria, irrespective of their
staining properties [41]. Moreover, the presence of a peri-
pheral layer containing PNA may not be a complete ex-
planation of the structure responsible for the Gram-staining

PLATE III. — Photographs of fixed cells of a colon bacillus stained by
the Giemsa technique: i, untreated cells; 2, fixed cells treated
with ribonuclease prior to staining; 3, treated with ribo-
nuclease and deoxyribonuclease. Ribonuclease removes cyto-
plasmic PNA responsible for the overall staining in i and
enables the presence of chromatinic (nuclear) bodies to be
shown (2). The latter are composed of DFNA and are not
present in the cells treated with deoxyribonuclease (3)

NUCLEOTIDES I33

reaction, since Gram-positive but not Gram-negative bac-
teria contain large amounts of the phosphates of various
polyalcohols, in particular, glycerol. If a suspension of Staph,
aureus is shaken with minute glass beads, the cells are dis-
rupted, but the outer layer of the cell — the 'cell wall' or 'cell
envelope' — remains intact, and it is with this that the major
portion of the polyol phosphates is associated [42]. However,
no direct evidence has yet been presented that these phos-
phates participate in the Gram-staining reaction. The recent
work of Bartholomew and Mittwer has provided further
support for the view that a layer immediately internal to
the cell wall is the site of the Gram-staining reaction [3].

The nuclei of plant and animal cells are rich in nucleic
acids, mostly of the DPNA type, and it is now generally
accepted that transmission of hereditable characters is
associated with these substances. Whether or not bacteria
possess a nucleus has been the subject of endless and incon-
clusive discussion, but they do undoubtedly contain struc-
tures composed of DPNA and known as chromatinic bodies.
The latter can be demonstrated in living cells by dark ground
phase contrast microscopy [50] and in dead cells by the
Feulgen or Giemsa staining technique. The Feulgen tech-
nique is based on the fact that after being subjected to acid
hydrolysis, deoxypentose nucleic acids, but not nucleic acids
of the pentose type, restore the colour of Schiff's reagent,
consequently cellular structures composed of DPNA be-
come stained magenta. Since any aldehyde is capable of
giving a positive reaction, the results should not be accepted
without confirmatory evidence. If an observed staining re-
action is due to PNA or DPNA, it should no longer be given
by material previously treated with the appropriate enzyme,
ribonuclease or deoxyribonuclease (p. 134), and then washed
(Plate III). Caution is also required in interpreting the re-
sults obtained in this manner since it may not be justifiable
to assume (i) that the enzyme can penetrate the experimental
material and thus come into contact with the substrate and
(ii) that the enzyme preparation is specific in its activity.

Although the evidence is often indirect, , there are good
reasons for believing that in micro-organisms, as in the

134 NITROGEN METABOLISM

more complex forms of life, the inheritance of specific char-
acters is controlled by the deoxypentose nucleic acids, and
in this respect a group of natural substances, known as trans-
forming factors, are of particular interest. A transforming
factor induces a susceptible cell to acquire a particular here-
ditable and characteristic property of the cell from which
the factor emanates. Once a cell has been 'transformed', the
acquired feature is transmitted through all subsequent
generations. The most thoroughly investigated example of
this phenomenon is provided by the pneumococci whose
virulence is associated with the possession of a capsule of
polysaccharide material. Differences in the composition of
the latter have enabled the pneumococci to be grouped into
more than thirty serologically distinct types. Griffith ob-
served that living non-encapsulated avirulent type II pneu-
mococci were changed into virulent encapsulated type III
pneumococci by passage of the former together with heat
killed cells of the latter through mice. Such transformations
can be brought about in vitro in certain well-defined condi-
tions and later Avery and his colleagues obtained convincing
evidence that the agents responsible for the transformation
of pneumococcal types were deoxypentose nucleic acids,
each acid being specific for one type of transformation
[see i]. More recently, other transformations dealing with
capsulation, resistance to penicillin and the ability to fer-
ment particular sugars have been demonstrated with certain
strains of Haem. influenzae, Esch. coli, Shigella paradysen-
teriae, B. anthracis and Pr. vulgaris; as in the pneumococci,
the factors accomplishing these transformations appear to be
deoxypentose nucleic acids [i]. Pentose nucleic acids have
been implicated in the formation of stretolysin S, the O2-
stable haemolytic exotoxin of Strep, haemolyticus [7].

Enzymic degradation of nucleic acids [31]

The enzymic degradation of nucleic acids commences
with disruption of the internucleotide linkages by ribo-
nuclease or deoxyribonuclease, enzymes specific for the
pentose and deoxypentose nucleic acids respectively: the
nucleic acid is thus reduced to a mixture of mono-, di- and

NUCLEOTIDES I35

a few oligo-nucleotides. Whilst the properties of the nu-
cleases of mammals and yeast have been studied in some
detail, the corresponding bacterial enzymes have received
comparatively little attention. Streptococcus haemolyticus
(Group A) secretes both types of nuclease into the
medium [34, 8], and ribonuclease is particularly active
during the initial stages of bacterial autolysis [cf. 23]. At
least as far as ribonuclease is concerned, depolymerization
is due to hydrolysis of the bond linking C'-5 of one nucleo-
tide to the phosphate group attached to C'-3 (or C'-2) of
an adjacent pyrimidine nucleotide. Analogous linkages be-
tween a pyrimidine and a purine nucleotide or between
two purine nucleotides are stable to ribonuclease, and the
enzyme may therefore be regarded as being a highly specific
phosphodiesterase [38]. Nuclease activity can be determined
by using a solution of the appropriate nucleic acid and fol-
lowing (i) changes in viscosity, (ii) the appearance of acid-
soluble phosphorus compounds, (iii) the appearance of
acidic groups or (iv) the decrease in the absorption of ultra-
violet light of wavelength 300 m/<.

After nucleotides have been dephosphorylated by appar-
ently non-specific phosphatases, the resultant nucleosides
may be attacked in one of three ways [28]. One type of
nucleosidase catalyses their phosphorolytic decomposition
to the free base and a pentose phosphate by transferring the
sugar moiety to inorganic orthophosphate (reaction a). The
phosphorolytic nucleosidases of Esch. colt exhibit specificity
with regard to the nitrogenous base in that they attack either
purine compounds or pyrimidine compounds, but not both,
and yet no specificity is shown towards the sugar part of the
substrates. Lactobacillus helveticus [33] and Esch. coli [28]
possess another type of transferase, specific for deoxyribo-
sides and utilizing not inorganic phosphate but a nitrogen-
ous base as the acceptor (reaction b).

(a) uracil-riboside+H3P04 ^^ uracil+ribose-i -phosphate
(6) uracil— deoxyriboside+thymine ^^

th>Tnin? —deoxyriboside + uracil

The hydrolytic decomposition of nucleosides to the free

136 NITROGEN METABOLISM

base and sugar in the complete absence of phosphate and
other nitrogenous bases has been observed with prepara-
tions from bakers' yeast [12] and Lb. pentosus [29].

The removal of amino groups from the nitrogenous bases
is accomplished by highly specific deaminases, but apart
from the cytosine deaminase of Esch. coli and yeast [cf. 14],
the adenosine deaminase oi Neurospora [35] and the cytidine
deaminase of brewers' yeast and Esch. coli [53], little is
known about the occurrence and properties of these enzymes
in micro-organisms.

Oxidation and fermentation of purines and pyrimidines

Application of the enrichment culture technique has led
to the isolation of organisms capable of utilizing purines and
pyrimidines as sole sources of carbon, nitrogen and energy.
Thus CI. cylindrosporum ferments uric acid to NH3, CO 2
and acetic acid, while CI. acidi urici, in addition to uric acid,
can also utilize xanthine, guanine and hypoxanthine [2].
Since neither of these species attacks allantoin, it has been
suggested that they degrade uric acid by a route diiferent
from that found in animal tissues, and there is some evidence
that glycine is an important intermediate in these fermenta-
tions. Unlike the Clostridia, Micrococcus aerogenes attacks
neither uric acid nor glycine, and lactic acid is the main
acidic end-product in the fermentation of adenine or gua-
nine [54]. On the other hand, Micrococcus lactilyticus is
unable to utilize adenine, guanine or uric acid but ferments
hypoxanthine and xanthine to H2, COg, NH3 and urea,
together with propionic and acetic acids [55]. A number of
aerobic bacteria (species of Nocardia, Corynehacterium^
Mycobacterium and an unidentified soil organism) obtain
carbon, nitrogen and energy by the oxidation of pyrimi-
dines [30, 52]. Uracil and thymine are both oxidized to
barbituric acid, which is then split into urea and malonic
acid: the urea is subsequently decomposed by urease into
CO 2 and NH3, but the further steps in the metabolism of
malonic acid are unknown. The oxidation of thymine pro-
ceeds by way of 5-methylbarbituric acid rather than by
direct demethylation to uracil.

NUCLEOTIDES I37

Synthesis of purines

Though the synthesis of the nitrogenous bases and the
pentose sugars is here, for convenience, considered separ-
ately, such a division does not imply that the synthesis of
nucleotides necessarily proceeds by the phosphorylation of
a nucleoside formed by the joining together of the pre-
formed base and the pentose sugar. There is some experi-
mental evidence that glycosidation of a precursor precedes
completion of the heterocyclic ring systems found in purines
and pyrimidines. Moreover, the possibility must always be
borne in mind that the routes of synthesis are not the same
in diiferent species. Experiments with substances labelled
with isotopes have revealed that Esch. coli and yeast resemble
mammals in that they synthesize purines and pyrimidines
from relatively simple precursors, namely CO 2, NH3, for-
mate and glycine. In the synthesis of guanine by yeast, C-4
is derived from CO 2, C-2 and C-8 from formate or sub-
stances which give rise to formate (e.g. serine, the methyl
group of methionine), C-6, C-5 and N-7 from the carboxyl,
methene and amino-N of glycine respectively, and the re-
maining nitrogen atoms from NH3 [20]. The utilization of
other compounds for purine synthesis is indirect and in-
volves their prior conversion to CO 2, NH3, glycine or for-
mate. Only one of the intermediates between these simple
precursors and the completed purine is known. When Esch.
coli is grown in the presence of sub-bacteriostatic concentra-
tions of sulphanilamide or the folic acid analogue N-10-
methylpteroylglutamic acid, an amine identified as 4-
amino-imidazole-5-carboxamide (AIC) accumulates in the
medium [49]. This substance only requires the addition of
one carbon atom (corresponding to position 2) to complete
the purine ring, and since it can be utilized by yeast,
Lh. arahinosus and purine auxotrophs of Esch. coli and
Ophiostoma, it is reasonable to suggest that it is a natural
intermediate in purine synthesis. If glycine, a known pre-
cursor in purine synthesis, is added to the medium, produc-
tion of the amine is increased [45], whereas it is reduced by
methionine, especially if trace amounts of PAB are present,
and also by vitamin B;^,- Moreover, in the absence of purines,

138 NITROGEN METABOLISM

B12, but not PAB or folic acid, enhanced the utiHzation of
AIC by mutants of Esch. coli exacting towards purines [6].
These resuhs are in accord with previous observations that
the methyl groups of methionine serve as a source of one
carbon units in intermediary metabolism and that they are
related to formate, a substance known to be a precursor of
purine carbon in position 2. Furthermore, Bj^g bas already
been implicated in the metabolism of labile methyl groups
in mammals, and it is to be noted that this growth factor
had no effect on the utilization of the formyl derivative of
AIC, 4-formamino-imidazole-5-carboxamide, by the Esch.
coli mutants. Incubation of AIC with Esch. coli resulted in
the formation of a substance, possibly a pentoside, which
was five times more effective than AIC itself in supporting
the growth of the purine auxotrophs [5]. Though the routes
by which the various purine bases are synthesized may be
quite distinct, it is most likely that they have several steps
in common, and it is possible that one is formed directly
from another. The interconvertibility of the purines shows
species variation; thus whilst guanine and adenine are freely
interconvertible in Esch. coli [27] and Lh. casei, yeast is only
able to convert adenine to guanine. On the other hand,
Tetrahymena gelei, a protozoon exacting towards guanine,
can change guanine into adenine [26].

CO NH COOH CO

NH2 C \ CH NH CH

II CH II I II

C y C.COOH CO C.COOH

/ \ / / \ /

NH2 N NH2 NH

Amino-imidazolecarboxamide Anmiofumaric acid Orotic acid

Synthesis of pyrimidines

Although the pyrimidine ring system is also part of that
present in purines, it is apparent that the synthesis of these
two groups of nitrogenous bases proceeds by entirely inde-
pendent routes. For example, in experiments with yeast,
carbon from formate or glycine was incorporated into

NUCLEOTIDES I39

guanine and adenine but not into uracil orcytosine. Further-
more, although carbon from isotopically labelled lactate was
found in both types of compound, the resultant distribution
of the isotope suggests that it entered the pyrimidines via
oxaloacetate, and the purines via glycine [20]. Certain pyri-
midine auxotrophs of Neurospora grow much better on
uridine or cytidine than on the free bases, and this again
suggests that the latter are not natural intermediates in
nucleoside synthesis. Some of the mutants used orotic acid
(uracil-6-carboxylic acid) to the same extent as uracil
itself [32], whilst others, for which this replacement was
not possible, accumulated orotic acid in the medium [39].
Though oxaloacetate, aminofumarate and aminofumaric
acid diamide supported the growth of two of the mutants,
they were only one tenth as effective as uracil. These and
other observations led Houlahan and Mitchell to propose
that in Neurospora, the biosynthetic sequence was: oxalo-
acetate — >- a-A^-pentosylaminofumaric acid diamide — >-
A — > B — > pyrimidine nucleoside: they also suggested that
orotic acid is not a true intermediate but is related to the
precursors A and B. In this connection it is interesting to
note that a glycoside of orotic acid, probably the riboside,
has been isolated from the mycelium of a uridine requiring
mutant of Neurospora, and it is possible that this substance
is a natural intermediate [43]. Incubation of Sac. cerevisiae
with isotopically labelled orotic acid results in the appear-
ance of the isotope in the uracil, but not the guanine, of the
nucleic acids. Orotic acid has also been implicated in the
synthesis of pyrimidines by streptococci, Lh. casei and Lh.
bulgaricus [16]; the orotic acid requirements of the latter
organism can be replaced by ureidosuccinic acid, a sub-
stance related to aminofumaric acid [56].

Synthesis of deoxyribose, ribose, nucleosides and nucleotides

A partially purified preparation of an aldolase obtained
from Esch. coli has been shown to condense glyceraldehyde-
3 -phosphate with acetaldehyde and thus form deoxyribose-
5-phosphate, which is converted by an isomerase, present
in crude extracts, to deoxyribose- 1 -phosphate [44]. Whilst

140 NITROGEN METABOLISM

ribose- 5 -phosphate may likewise be synthesized from
phosphoglyceraldehyde and glycolaldehyde, Esch. colt and
Sac. cerevisiae are known to be capable of performing
the series of reactions, glucose — > glucose-6-phosphate — >-
6-phosphogluconate — > 3(2)-ketogluconic acid — > ribulose-
5-phosphate — > ribose-5-phosphate — > ribose- 1 -phosphate
[22, 36]. In view of the reversible nature of the nucleosidases,
it is reasonable to suggest that transfer reactions between
the pentose- 1 -phosphates and purines or pyrimidines are a
possible route of nucleoside synthesis. There is, however,
no proof that this is the natural pathway and mention has
already been made of some evidence, admittedly indirect,
which indicates that glycosidation precedes completion of
the heterocylic rings present in purines and pyrimidines.
With regard to the phosphorylation of nucleosides to form
nucleotides, all of our present knowledge concerns nucleo-
tides known to be constituents of coenzymes rather than of
nucleic acids. The following syntheses, catalysed by enzyme
preparations obtained from yeast {c, d, f) and from liver
\e, g) have been described [24, 46]:

(c) adenosine+ATP — > adenosine-5-phosphate+ADP

Id) riboflavin+ATP — > FMN+ADP

{e) nicotinamide-riboside+ATP — >■

nicotinamide-riboside-5-phosphate+ADP
(/) FMN+ATP — > FADN+pyrophosphate

(^) Nicotinamide-riboside-5-phosphate+ATP — >

DPN +pyrophosphate

Effect of bacteriophage on nucleic acid metabolism of host
cell [17]
All viruses consist essentially of nucleic acids and pro-
teins, and, in addition, the more complex types infecting
animals also contain fatty material. Deoxypentose nucleic
acids and PNA are found respectively in bacteriophages and
plant viruses, and although most animal viruses contain only
one type of nucleic acid, a few, e.g. the influenza virus,
appear to contain both DPNA and PNA. When purified
fully infective preparations of various viruses have been
examined for the presence of known enzymes, no activity

NUCLEOTIDES I4I

has been found except in those of the more complex animal
viruses. Interest has therefore centred around the mechan-
ism whereby a virus assumes control of the metabolic
activities of the host cell and reorganizes them for the syn-
thesis of identical virus particles. The virus-host-cell system
investigated in most detail is that concerned with the infec-
tion of Esch. coli B with a phage designated as the T phage,
of which seven types are known. Electron microscopy has
revealed that Ti and T5 are more or less spherical, whereas
the other five T phages are club-shaped and have a distinct
head, containing most of the DPNA, and a tail. Phage repro-
duction involves three phases: (i) adsorption on to and
invasion of the susceptible cell; (ii) multiplication in the host
cell, and (iii) liberation from the host cell, a process usually
accompanied by lysis, though liberation and lysis are not
always coincident. Unless stated to the contrary, the follow-
ing description applies to phage T2 and the details are not
necessarily the same for other viruses.

The adsorption of T2 by Esch. coli B is a rapid and
reversible process dependent on the presence of tryptophan
in the medium. It is soon followed by an irreversible process
in which part of the virus, mainly DPNA and a little pro-
tein, enters the cell, an event which immediately results in
a marked disturbance of the host cell's nucleic acid meta-
bolism, the first visible signs being degradation of the
chromatinic bodies. The inability of virus-infected cells to
grow and divide is presumably an outw^ard expression of
this destruction of nuclear material. Nucleic acid synthesis
is at first completely halted, but after a little while the
synthesis of purines and pyrimidines commences and is soon
followed by the formation of DPNA. Pentose nucleic acid
synthesis is completely suppressed in Esch. coli B infected
with T2, but in other virus-host-cell systems, the synthesis
of both types of nucleic acid may take place. The amount
of DPNA synthesized is equivalent to the total amount of
both types of nucleic acid synthesized by uninfected cells,
and this indicates that in the infected cells the units
normally incorporated into PNA are being diverted to the
synthesis of DPNA. In attempting to discover the reason

142 NITROGEN METABOLISM

for this diversion Cohen has found that infection with phage
causes a marked reduction in the abiUty of the cell to form
ribose-5-phosphate from glucose-6-phosphate (p. 140), and
this presumably may explain why such cells are unable to
synthesize RNA. The synthesis of deoxyribose- 5 -phosphate
from glucose-6-phosphate is unimpaired and consequently
deoxypentose-nucleotide synthesis is unaffected.

Infection with phage has no visible effect on protein
synthesis in the host cell, and although the amount of pro-
tein synthesized is in excess of that found in the number of
virus particles eventually liberated, it is not yet known
whether all the excess is viral protein. The origin of the
various substances present in the phage has been determined
by using cells whose cellular constituents have been enriched
with isotopes and also by suspending the infected cells in
media containing the appropriate compounds labelled with
isotopes. As far as T2 is concerned, no more than 25% of
the phosphorus, protein nitrogen, pyrimidines and purines
in the liberated phage is derived from substances present in
the host cell at the time of infection. How the virus particle
gains control of the anabolic activities of the host cell is
still unknown. It is however clear that the term 'self-
duplicating particle' should not be used indiscriminately
since, for example, the synthesis of protein and nucleic acid
in infected cells proceeds at a linear rate, whereas if the
system were self-duplicating, one might expect it to be auto-
catal5rtic. Moreover, there is evidence that the virus is
radically changed during invasion of the host cell, since
much of the phosphorus and some of the protein nitrogen
of the infecting phage particle soon appears in the medium,
and not for an appreciable time after invasion is it possible
to isolate infective virus from the host cell. The assembling
of the various units into completed virus appears to occur
only a short time prior to liberation. In the T2 system, the
first recognizable structures to be found in the infected cells
are the collapsed heads of the phage containing only a little
DPNA, the rest of the latter and the tails being added later.

NUCLEOTIDES I43

REFERENCES

1. Austrian, R. (1952), Bad. Rev., 16, 31

2. Barker, H. A. and Beck, J. V. (1942), J?^. Bact., 43, 291 '

3. Bartholomew, J. W. and Mittwer, T. (1952), Biol. Rev., 16, i

4. Belozersky, A. N. (1947), Cold Spr. Harb. Symp., 12, i

5. Ben-Ishai, R., Bergmann, E. D. and Volcani, B. E. (1951),

Nature, 168, 11 24

6. Bergmann, E. D., Volcani, B. E. and Ben-Ishai, R. (1952),^.

hiol. Chem., 194, 521, 53 1

7. Bernheimer, A. W. (1949), jf. exp. Med., 90, 373

8. (1953), Biochem. J., 53, 53

9. Caputto, R., Leloir, L. F., Cardini, C. E. and Paladini, A. C.

(1950), J' biol. Chem., 184, 333

10. Caspersson, T. (1947), Soc. exp. Biol. Symp., i, 127

11. Carter, C. E. (1950), j^. Amer. chem. Soc, 72, 1466

12. (i95i)> J- Amer. chem. Soc, 73, 1508

13. Chargaff, E. (195 1), Fed. Proc, 10, 654

14. and Kream, J. (1948),^. hiol. Chem., 175, 993

15. and Saidel, H. F. (1949), J. biol. Chem., 177, 417

16. Christman, A. A. (1952), Physiol. Rev., 32, 303

17. Cohen, S. S. (1951), Bad. Rev., 15, 131

18. Cohn, W. E. (1950),^. Amer. chem. Soc, 72, 1471

19. and Carter, C. E. (1950), J. Amer. chem. Soc, 72, 2606

20. Edmunds, M., Delluva, A. M. and Wilson, D. W. (1952), J.

biol. Chem., 197, 251

21. Henr\', H. and Stacey, M. (1946), Proc. Roy. Soc, 133B, 391

22. Horecker, B. L. and Smyrniotis, P. Z. (195 1), J. biol. Che7?i.,

I93» 371, 383

23. Jones, A. S., Stacey, M. and Webb, M. (1949), Biochim.

Biophys. Acta, 3, 383

24. Kearney, E. B. and Englard, S. (1951),^^. biol. Chem., 193, 821

25. Kerr, S. E., Seraidarian, K. and Wargon, M. (1949), J. biol.

Chem., 181, 761

26. Kidder, G. W. and Dewey, V. C. (1948), Proc Nat. Acad. Sci.,

34, 566

27. Koch, A. L., Putnam, F. W. and Evans, E, A. (1952), jf. biol.

Chem., 197, 105

28. Lampen, J. O. (1952), Bad. Rev., 16, 211

29. and Wang, T. P. (1952), J. biol. Chem., 198, 385

30. Lara, F. J. S. (1952), J. Bact., 64, 271, 279

31. Laskowski, M. in The Enzymes, 1 (ii), Chap. 29

32. Loring, H. S. and Pierce, J. G. (1944), J- ^iol. Chem., 153, 61

33. MacNutt, W. S. (1952), Biochem. y., 50, 384

34. McCartv, M. (1948),^. exp. Med., 88, 181

35. McElrov, W. D. and Mitchell, H. K. (1946), Fed. Proc, 5, 376

36. McNair-Scott, D. B. and Cohen, S. S. (1951), J. biol. Chem.,

144 NITROGEN METABOLISM

37. Markham, R. and Smith, J. D. (1949), Biochem. J., 45, 294
38. (1952), Biochem. y., 52, 552, 558, 565

39. Mitchell, H. K., Houlahan, M. B. and Nye, J^ F. (1948), J.

biol. Chem., 172, 525

40. Mitehell, P. {1950), jf. gen. Microbiol., 4, 399

41. and Moyle, J. (1950), Nature, 166, 218

42. and Moyle, J. (195 1), jf. gen. Microbiol., 5, 966, 981

43. Mitchelson, A. M., Drell, W. and Mitchell, H. K. (195 1),

Proc. Nat. Acad. Sci., 37, 396

44. Racker, E. (1952), J. biol. Chem., 196, 347

45. Ravel, J. M., Eakin, R. E. and Shive, W. (1948), J', biol. Chem.,

172, 67

46. Rowen, J. W. and Romberg, A. {1951), J- biol. Chem., 193, 497

47. Schmidt, G. and Thannhauser, S. J. (1945), jf. biol. Chem.,

161, 83

48. Schneider, W. C. (1945), 3^- biol. Chem., 161, 293

49. Shive, W., Ackermann, W. W., Gordon, M., Getzendaner,

M. E. and Eakin, R. E. (1947), jf. Amer. chem. Soc, 69, 725

50. Stempen, H. (1950),^. Bad., 60, 81

51. Vischer, E., Zamenhof, S. and Chargaff, E. (1949), jf. biol.

Chem., 177, 429

52. Wang, T. P. and Lampen, J. O. (1952), jf. biol. Chem., 194,

775, 785

53. Sable, H. Z. and Lampen, J. O. (1950), jf. biol. Chem.,

184, 17

54. Whiteley, H. R. (1952),^. BacL, 63, 163

55. and Douglas, H. C. (195 1), jf. Bact., 61, 605

56. Wright, L. D., Miller, C. S., Skeggs, H. R., Huff, J. W.,

Weed, L. L. and Wilson, D. W. (195 1), J. Amer. chem. Soc,
73, 1898

57. Wvatt, G. R. (1951), Biochem. J., 48, 581, 584

CHAPTER X

MODE OF ACTION OF
CHEMOTHERAPEUTIC AGENTS

In recent years, the dramatic success of the sulphonamides
and antibiotics in the treatment of bacterial and viral infec-
tions of man has focused much attention on the mode of
action of these substances and the factors contributing to
their activity. Substances interfering with the continued
normal existence of an organism may owe their properties
to (i) denaturation of proteins, (ii) disruption of cellular
membranes and in consequence the leakage of essential
metabolites, or (iii) more specifically, inhibition of particular
enzymes. Substances bringing about the first two of these
effects are of little chemotherapeutic importance, because
of their adverse effect on the host as well as the pathogen.
The key to successful chemotherapy is contained in the
phrase selective toxicity, i.e. the drug is more toxic to the
pathogen than to the host. If a chemotherapeutic agent acts
by inhibiting an enzymic system of fundamental importance
to the pathogen, then this system must be absent from the
host, or, if present, it is for some reason less susceptible to
the drug, or not essential. (For a detailed exposition of the
principles of chemotherapy and selective toxicity, references
31 and I are recommended.) One of the aims of contem-
porary research is to establish a rational basis for chemo-
therapy, and with this in mind, the purpose of the following
paragraphs is to survey very briefly the observed effects
of the sulphonamides and antibiotics on the metabolism of
nitrogenous compounds.

It must be emphasized that great care is required in
deciding whether the observed effect produced by a drug is
due to direct inhibition of the reaction leading to that end
result, or whether it is a secondary effect arising out of the
inhibition of some other, perhaps unknown, reaction. Even
if the drug inhibits cell-free preparations of an enzyme this

145

146 NITROGEN METABOLISM

does not constitute proof that its mode of action against the
intact organism is necessarily expUcable in such terms.
Before the latter can be attempted, precise and quantitative
information is required deaUng with the significance of that
enzyme in the general economy of the cell (e.g. turnover
numbers). Moreover, ideally the observed results should be
produced by drug concentrations of the same order as those
used therapeutically. But, since the concentration of cells
in washed suspension experiments is often many times
greater than those in growing cultures or infected animals,
it has been argued that this proviso can be ignored [14];
furthermore, it is also feasible that the drug enters growing
and dividing cells more readily than resting cells [10].

Sulphonamides

The first major contribution towards understanding the
mode of action of the sulphonamides was made by Woods
who prepared from yeast an aromatic carboxylic amine
which competitively antagonized the action of sulphanila-
mide in preventing the growth of Strep, haemolyticus. Fildes'
suggestion that a chemotherapeutic agent might function
by virtue of its chemical structure being such that it was
adsorbed on to an enzyme in place of the natural substrate
led Woods to infer that the isolated material was ^-amino-
benzoic acid (PAB) and he showed that the latter did in fact
antagonize the sulphonamides in a competitive manner [28].
In other words, the biological activity of the sulphonamides
could be explained on the grounds that they were non-
utilizable analogues of a natural metabolite, namely, PAB.
Until that time the importance of PAB in intermediary
metabolism had not been suspected, but evidence soon
became available that PAB was a growth factor for certain
organisms, and that a group of substances containing PAB
— the folic acid factors required by certain other organisms
— ^were of universal importance. The various folic acid
factors differ in the number of glutamic acid radicals in the
molecule, the degree of reduction of the pterin and the
presence or absence of a formyl group attached to one of
the nitrogen atoms of the pterin or PAB. For example, the

CHEMOTHERAPEUTIC AGENTS I47

*folic acid' requirements (citrovorum factor or folinic acid)
of Leuconostoc citrovorum are replaceable by a synthetic
material, A^-5-formyl-5, 6, 7, 8-tetrahydropteroylglutamic
acid, but not by pteroylglutamic acid itself (synthetic folic
acid). The experimental evidence favours the view that
organisms utilize PAB solely for the synthesis of 'folic acid'
but the detailed constitution of the natural coenzyme con-
taining combined PAB is still unknown [29].

The role of PAB in intermediary metabolism was ex-
plored further by a comprehensive survey of the substances
other than folic acid which were capable of antagonizing the
growth inhibitory action of the sulphonamides. This proce-
dure is based on the hypothesis that if PAB or a derivative
functions catalytically in the synthesis of substances essential
for growth, and if the sulphonamides act by preventing the
normal functioning of PAB, then growth should be resumed
if these substances are supplied exogenously. In a sense
such cultures can be regarded as being deficient in PAB
or folic acid, and the principles involved are the same as
in the growth factor replacement technique in which an
attempt is made to replace a grov^rth factor either by simpler
substances from which it can be synthesized, or by sub-
stances whose synthesis the growth factor is suspected to
mediate. Apart from folic acid, the natural antagonists of
the sulphonamides fall into three groups, (i) amino-acids,
in particular methionine and serine, (ii) purines such as
xanthine and (iii) thymine and thymidine [22, 27]. For
example, the addition of methionine to the medium
decreased the amount of PAB required to overcome the
inhibitory effects of sulphanilamide on the growth of Esch.
colt. The PAB requirement was further reduced if xanthine
was also included and diminished still further, if the
medium contained methionine, xanthine and serine. It was
abolished altogether when thymine was added in addition
to these three substances. Similarly with a PAB auxotroph
of Esch. coll growth was possible in the absence of PAB,
provided the medium contained methionine, a purine and
thymine. Analogous experiments have been done with
other organisms and the same three groups of substances

148 NITROGEN METABOLISM

are also active in antagonizing the growth inhibitory pro-
perties of analogues of folic acid, e.g. A:-methylfolic acid.

Previous work with animal tissues and Neurospora indi-
cated that serine was synthesized by the addition of formate

p-aminobenzoic acid

HOOC

precursors

glycine + formate

f

serine

precursors

valine

lysine

threonine

histidine

homocysteine
methionine

H©

deoxyribosides

of guanine, adenine

and cytosine

citrovorum

factor

(f clinic acid)

combined PAB
Folic acid ?

CHzCOOH

ch.nh.oc-^~\nh .CH2-«|

COOH

pteroylglutamic acid
(synthetic folic acid)

-t

»""=

precursors

thymidine
thymine

precursors

amino-
imidazole
carboxamide

MS)

vitamin
^2

purines

FIG. 10. 1. — Role of ^-aminobenzoic acid and vitamin B12 in
the synthesis of amino-acids, purines and pyrimidines

to glycine, and since glycine, unlike serine, did not antagonize
sulphanilamide, it was concluded that the latter was an
inhibitor of this synthesis. Woods and his colleagues have
now shown that though Ln. mesenteroides is exacting towards
serine, growth occurred in the absence of the amino-acid

CHEMOTHERAPEUTIC AGENTS I49

provided pyridoxin, PAB, CO 2 and glycine were available.
Further evidence that pyridoxin as well as PAB partici-
pated in the synthesis of serine from glycine was obtained by
using cells grown in the absence of these two growth factors
in a medium containing all the other known grov^h fac-
tors together with amino-acids, purines and pyrimidines.
Washed suspensions of these cells would synthesize serine
only if they were provided with glycine, formate, glucose,
pyridoxin and PAB (or folinic acid); synthesis was com-
pletely inhibited by sulphanilamide. Similar experiments
have been done with Strep. faecaliSy Lb. hifidus and Sac.
cerevisiae. It appears that both PAB and vitamin B^g play
a role in the synthesis of methionine, the most active amino-
acid antagonist of the sulphonamides. A vitamin B^2 auxo-
troph of Esch. coli grew in the absence of B^g if the medium
contained methionine [4], while cells of a mutant requiring
PAB and grown in the absence of this factor (i.e. in a
medium containing amino-acids, purines and pyrimidines)
only synthesized methionine from homocysteine in the
presence of PAB and glucose. The simultaneous addition of
B12 stimulated synthesis threefold, and recent work suggests
that the methyl group used in the methylation is derived
from serine. There is some evidence that the role of PAB
in methionine synthesis cannot be explained solely on the
basis that it is required for the synthesis of B12 and
serine [29].

The connection between PAB and the synthesis of the
group of substances now designated as vitamin B12 (the
cobalamins) began with the observation that the anaemia
produced by feeding an animal large amounts of sulpha-
thiazole was like pernicious anaemia and could be relieved
by large doses of synthetic folic acid or concentrates of sub-
stances isolated from the liver of normal animals. These
liver substances were not of the folic acid type and functioned
as growth factors for Lb. leichmannii and Lb. lactis. The key
compound, vitamin B^g, active both as a growth factor and in
the treatment of pernicious anaemia, has now been iso-
lated in the crystalline state and is composed of 5:6-di-
methylbenzimidazole - 1 - a - D -ribofuranoside-3 -phosphate

150 NITROGEN METABOLISM

combined with an unidentified organic molecule contain-
ing cobalt. Though the B^g requirements of a micro-organ-
ism cannot be replaced by any of the known folic acids,
either synthetic or natural, they are replaceable by thymi-
dine and also in most organisms, by the deoxyribosides of
purines. This is to be contrasted with the fact that though
thymidine replaces part of the PAB or folic acid require-
ments of an organism, all other deoxyribosides are inactive.
Vitamin B^g contains an aromatic ring, yet it did not stimu-
late the growth of mutants with a multiple requirement for
aromatic compounds. Davis therefore suggested that it was
either derived from one of the aromatic substances required
by such mutants or else it was synthesized by a totally
different route or from an intermediate prior to the
genetically blocked reaction. Vitamin B^g exerts a sparing
effect on the PAB requirement of a PAB auxotroph of Esch.
coli, and since the amount of vitamin B12 required is only
one fiftieth of the amount of PAB, it is conceivable that the
ring of PAB is used directly in the synthesis of B^g [3]. On
the other hand, folic acid is known to be associated with the
synthesis of purines, which, like B^gj contain an imidazole
ring, and this may be the reason for the close relationship
between these two co-factors. At the present time, the
details of the relationship are but vaguely understood and
have only been explored in one species, Esch. coli.

From these and other studies, it has become evident that
the ultimate co-factor form of PAB participates in the
methylation of homocysteine, the introduction of carbon
into position 2 of the purine ring (pp. 1 37-8), and the synthesis
of serine and thymine; in other words, this co-factor is con-
cerned with the intermediary metabolism of one carbon
units (cf. Co.A, the ultimate co-factor form of pantothenic
acid, and its function in the metabolism of acyl units [20]).
There is also reason to believe that Bjg is involved in at
least some of these reactions, e.g. the synthesis of methionine
and purines, and it is to be noted that the presence of B^g
increases the amount of sulphanilamide required to induce
bacteriostasis of Esch. coli growing in the presence of one
or more of the sulphonamide antagonists methionine,

CHEMOTHERAPEUTIC AGENTS I5I

xanthine, serine and thymine [21]. Other effects of the
sulphonamides have been reported, but whether they are
also the outcome of interference with reactions involving
PAB is not always known, e.g. sulphathiazole appears to
interfere with protein synthesis in Staph, aureus, but since
protein synthesis involves a complex metabolic sequence of
reactions, it is not possible to deduce whether this is a
direct effect or simply due to disturbances in the synthesis
of amino-acids or nucleotides [5]. It has been shown that
the even- but not the odd-numbered T phages are unable to
multiply in Esch. coli growing in media containing sul-
phanilamide together with methionine, serine, xanthine and
thymidine [19]. The results of these and other experiments
require to be interpreted with care, e.g. Pfiffner and his co-
workers have isolated from bacteria compounds of the
vitamin B^g group which contain adenine instead of di-
methylbenzimidazole, and Davis later found that these
substances (pseudo-Big) replaced B^g i^i all respects for
Esch. coli mutants requiring this vitamin or methionine.

Antibiotics

Though the precise details are still unknown, it is highly
probable that the sulphonamides owe their activity to the
fact that they are metabolic analogues. On the other hand,
no such simple hypothesis is available to explain the activity
of any of the antibiotics. There is no conclusive evidence
that penicillin inhibits enzyme systems concerned in respira-
tion or fermentation, and it is therefore unlikely that any
of the results described below are attributable to direct
interference with energy metabolism. By way of contrast,
aureomycin resembles 2:4-dinitrophenol and sodium azide
in that it may act as an uncoupling agent and prevent the
production of energy-rich phosphate groups [15]. If peni-
cillin (o-i-io Oxford Units /ml.) was added to growing
cultures of Staph, aureus, then within an hour of contact
with the antibiotic the ability of the organism to accumulate
amino-acids and synthesize protein progressively declined
[8]. Penicillin had no effect on the uptake, of glutamic acid
and lysine by washed suspensions of normal cells, hence it
II

152 NITROGEN METABOLISM

would appear that this antibiotic prevents the synthesis of
the systems responsible for the absorption of amino-acids
and does not affect the functioning of those systems once
they have been established. When washed suspensions of
cells grown for a short time with penicillin were incubated
with glucose and glutamic acid, little of the latter accumu-
lated in the cells, though extracellular peptides of glutamic
acid appeared in the system [7]. These results are analo-
gous to those of Hotchkiss, who used a different species
of staphylococcus and different experimental conditions.
Washed suspensions incubated with a mixture of various
amino-acids and glucose synthesized protein, but in the
presence of penicillin there was no increase in cellular com-
bined amino-acids though the number of free amino-groups
in the medium decreased. As in Gale's experiments, the
latter was correlated with the appearance of extracellular
peptides, and Hotchkiss suggested that penicillin inhibited
protein synthesis and that these peptides were either inter-
mediates in this process or were derived from them [13].
Nevertheless, it is difficult to believe that penicillin inter-
feres directly with the synthesis of all proteins since it has
no effect on the formation of adaptive enzymes, a process
now regarded as being associated with the synthesis of new
protein. Streptomycin, aureomycin, chloramphenicol and
terramycin inhibit adaptive enzyme formation [11], an effect
which is possibly the outcome of interference with energy
metabolism.

By examining a number of strains of Staph, aureus^ Gale
found that increased resistance to penicillin could be
correlated with a decline in ability to accumulate glutamic
acid. It will be recalled that Gram-negative, unlike Gram-
positive, organisms do not concentrate amino-acids in the
cells (p. 82), and the most highly resistant variants of
Staph, aureus obtained by successive subculture in increas-
ing concentrations of penicillin were in fact Gram negative.
Moreover, these organisms were no longer cocci but rod-
shaped and had lost the ability to utilize certain sugars and
grow anaerobically. Several workers have noted one or more
of these effects (i.e. changes in morphology. Gram-staining

CHEMOTHERAPEUTIC AGENTS 153

properties and ability to metabolize various substances, and
also a preference for aerobic growth) with other species
growing in the presence of penicillin. By studying whether
the development of resistance is accompanied by overall
changes in the metabolism of the organism, it may be
possible to gain valuable information concerning the mode
of action of the agent being considered, and furthermore,
if resistance to other drugs is acquired simultaneously, i.e.
cross resistance, it is conceivable that the biological effects
of all these substances is explicable in the same terms [see 31].
Another as yet unexplained observation that penicillin inter-
feres with amino-acid metabolism concerns an unidentified
Gram-negative organism which when growing on L-leucyl-
glycine in a mineral salt medium was relatively insensitive to
penicillin. Leucine and glycine, either singly or together,
also supported growth, but in the presence of uncombined
glycine the organism was very sensitive to penicillin (i to
10 units/ml.) [23].

The reports that the training of Staph, aureus to a high
degree of resistance to penicillin resulted in a loss of Gram-
positive staining properties could be taken to indicate that
the biological effects of penicillin were the outcome of
primary disturbances in nucleotide metabolism. In normal
cultures of Staph, aureus the rate of cell growth appears to
be controlled by the amount of pentose nucleic acid in the
cells, and the cellular concentration of soluble nucleotides
is inversely proportional to the rate of PNA synthesis [17]. If
penicillin is added to a culture in the log phase of growth,
then before there is any visible change in the growth-rate,
the concentration of soluble nucleotides increases and the
ratio of soluble nucleotides to total nucleic acid soon changes
from o-i to 0-2 (Fig. 10.2). The percentage by weight of
nucleic acid at first appears to increase, not because synthesis
is stimulated but because there is a decrease in the rate of
synthesis of some other substances (protein?) contributing
to the dry weight of the cell. Though large amounts of
nucleic acid are normally present in young cells, penicillin
causes their concentration to fall rapidly to the low value
characteristic of old cells in the stationary phase of growth,

154

NITROGEN METABOLISM

Q ■ • NORMAL CULTURE

•-C-- CULTURE PLUS

\fj^. PENICILLIN/ML.

8 20 22

T 1 I I n T

6 8 10 12 14 16
TIME (in hours)

FIG. I0.2. — Effect of penicillin on nucleic acid metabolism of Staph,
aureus. %NA=percentage by weight of total nucleotide,
%Nt= percentage by weight of nucleotide extractable with
5% trichloracetic acid, NA-Nt=total nucleic acid, DW— dry
weight of organism in /xg./ml. [17]

CHEMOTHERAPEUTIC AGENTS 155

and the ability to adsorb glutamic acid is not impaired until
this process is taking place. From such results it may be
deduced that penicillin interferes not with nucleotide syn-
thesis but with their polymerization to nucleic acid. Park
and Johnson have noted that the gro\sth of Staph, aureus
in the presence of penicillin (i unit/ml.) leads to the accu-
mulation of intracellular compounds containing uridine-
5 -pyrophosphate and an unidentified amino carboxylic
sugar: a peptide of D-glutamic acid, D-valine and DL-lysine
is a component of one of these compounds, whilst another
contains L-alanine [i8]. Synthesis of such nucleotides only
occurs for as long as the cells are viable, and Park suggests
that they are natural intermediates whose utilization is
inhibited by penicillin. Maass and Johnson have shown that
part of the penicillin absorbed by a cell is irreversibly bound
within it, and they postulate that the antibiotic combines
with and thus inhibits the natural functioning of a cellular
constituent which is normally present in small amounts and
controls the processes of cell division [16].

Penicillin is lethal to most Gram-positive organisms and
is effective against only a few Gram-negative species,
whereas streptomycin, chloramphenicol and aureomycin are
active against a wide variety of organisms, and the two latter
are also valuable in the treatment of diseases due to viruses
and rickettsiae. Streptomycin had no effect on the accumu-
lation of glutamic acid by Staph, aureus, but in concentra-
tions markedly greater than those inhibiting growth, pre-
vented protein synthesis. Aureomycin and chloramphenicol
inhibited the absorption of glutamic acid and protein syn-
thesis, the latter being especially sensitive [6, 7]. Of the
many enzyme systems examined, only the diamine oxidase
activity of whole cells of Mycobacteria, Ps. aeruginosa and
Staph, aureus was inhibited by streptomycin, and there was
some evidence that inhibition of this oxidase resulted in the
cessation of growth [32]. Streptomycin contains basic
guanidine groups and a possible explanation was that it was
absorbed on to the enzyme in place of the natural basic
substrate. However, cell-free preparations of the oxidase
were but little affected by streptomycin, hence the observed

156 NITROGEN METABOLISM

result is not due to direct inhibition of this enzyme [9]. The
oxidase activity of whole cells of streptomycin resistant
variants of Myc. smegmatis was very much less sensitive and
like the mammalian enzyme, only inhibited by high concen-
trations [32]. Other experiments indicate that this antibiotic
interferes with the entry of pyruvate into a terminal
pathway responsible for its oxidation [12, 25], and that this
pathway does not involve conversion to acetate and con-
densation of acetate with oxaloacetate to form citrate.
Streptomycin-resistant strains of Esch. coli do not possess
this pathway, and although it is also present in mammalian
mitochondria, permeability barriers prevent streptomycin
from having any effect [26]. Chloramphenicol inhibits
esterases in the cell-free state, but had no effect on forty
other enzymes examined [24]. There is some evidence that
it interferes with the metabolism of aromatic amino-acids,
e.g. with Esch. coli, the addition of phenylalanine, tyrosine
or tryptophan overcame the growth inhibitory effects of
low concentrations of chloramphenicol [30], and in Esch.
coli, as in Salm. typhosa, it appears to prevent the conversion
of anthranilic acid to indole [2].

Although the above account is very incomplete, it serves
to illustrate that much has still to be discovered before a
precise statement can be made concerning the mode of
action of the sulphonamides and antibiotics. Nevertheless,
apart from their potential value in the development of new
chemotherapeutic agents, such studies have made and can
make valuable contributions to the general pool of bio-
chemical knowledge.

REFERENCES

1. Albert, A. (1951), Selective Toxicity, Methuen, G.B.

2. Bergmann, E. D. and Sicher, S. (1952), Nature, 170, 931

3. Davis, B. D. (1951), 3^. Bact., 62, 221

4. and Mingiolo, E. S. (1950), J. Bad., 60, 17

5. Gale, E. F. (1947),^. gen. Microbiol, i, 327

6. and Folkes, J, P. (1953), Biochem. J., 53, 493

7. and Paine, T. F. (195 1), Biochem. J., 48, 298

8. and Taylor, E. S. (1947), J. gefi. Microbiol., i, 314

9. Geronimus, L. H. (195 1), Bad. Proc, 128

CHEMOTHERAPEUTIC AGENTS I57

10. Gros, F., Beljanski, M. and Macheboeuf, M. (1951), Bull. Soc.

Chim. Biol., Paris, 33, 1696

11. Hahn, F. E. and Wisseman, C. L. (195 1), Proc. Soc. exp. Biol.

Med., 76, 533

12. Henry, J., Henr}% R. J., Housewright, R. D. and Berkman, S.

(1948), 7 Bact., 56^527

13. Hotchkiss, R. D. (1950), 3^. exp. Med., 91, 351

14. Krampitz, L. O. and Werkman, C. H. (1947), Arch. Biochem.,

12, 57

15. Loomis, W. F. (1950), Science, iii, 474

16. Maass, E. A. and Johnson, M. J. (1949), J. Bad., 58, 361

17. Mitchell, P. (1949), Nature, 164, 259

18. Park, J. T. (1952), J. biol. Chem., 194, 877, 885, 897

19. Rutten, F. J., Winkler, K. C. and de Haan, P. G. (1950), Brit.

y. exp. Path., 31, 369

20. Shive, W. (1950), Ann. N.Y. Acad. Set., 52, 1212

21. (1951), Vitamins and Hormones, 9, 75

22. and Roberts, E. C. (1946), jf. biol. Chem., 162, 463

23. Simmonds, S. and Fruton, J. S. (1950), Science, iii, 329

24. Smith, G. N., Worrel, C. S. and Swanson, A. L. (1949), J.

Bact., 58, 803

25. Umbreit, W. W., Smith, P. H. and Oginsky, E. L. (195 1), J.

Bact., 61, 595

26. and Tonhazy, N. E. (1949), J. Bact., 58, 769

27. Winkler, K. C. and de Haan, P. G. (1948), Arch. Biochem., 18,

97

28. Woods, D. D. (1940), Brit.y. exp. Path., 21, 74

29. (1952), Internat. Congress of Biochem., Microbial Meta-
bolism, p. 86

30. Woolley, D. W. {1950), J. biol. Chem., 185, 293

31. Work, T. S. and Work, E. (1948), The Basis of Chemotherapy,

Oliver and Boyd, G.B.

32. Zeller, E. A., Owen, C. A. and Karlson, A. G. (195 1), J. biol.

Chem., 188, 623

INDEX

Acetylsulphanilamide,synthesis,i03
Adaptation, 13
Adenine, deamination, 136
— , in nucleic acids, 126-7
Adenosine, deamination, 136
— , triphosphate, 3
Adenyhc acid, co-factor for deami-
nases, 23-5
D- Alanine and Lb. casei, loi
— , replacement of pyridoxin, 28,

lOI

L-Alanine, as H-donor, 17, 18, 21

— , deamination, 11, 17

— , fermentation, 21

— , from kynurenine, 14

— , racemase, 95

— , transamination, 60-4

Alcohols from amino-acids, 22

Aldolase, 139

Algae, nitrogen-fixation, 50

Amines, from amino-acids, 27

D-Amino-acids, in antibiotics, 96-7

— , in capsules, 96

- — , in peptides, 96-7, 155

— , oxidase, 11

— , racemase, 95

— , utilization, 95

L- Amino-acids, acetylation, 104

— , active transport of, 91

-^, antagonism, 65

— , as H-acceptors, 18

— , as H-donors, 17

— , as sources of carbon and nitro-
gen, 20-2

— , assay by decarboxylases, 28

— , decarboxylases, 27-9

— , fermentation, 17-22

— , free in Gram-positive bac-
teria, 82

— , in bacterial proteins, 95

— , oxidase, lo-ii

— , synthesis, 60-78

— , transamination, 60-4

— , uptake, 80-93

— , — , by Staph, aureus^ 86-9

— , — , by Strep, faecalis, 83-9

— , — , by yeast, 89

— , — , glutamic acid, 86-9, 91-3

— , — , lysine, 83-6, 90-1

— , — , mechanism, 89

^-Aminobenzoic acid, in folic acid,
146

, sulphonamide antagonist, 146

Aminofumaric acid, 139

, diamide, 139

4-Aminoimidazole-5-carboxamide,

137-8
Aminopolypeptidases, 113
Ammonia, and biotin-deficient

yeast, 25
— , as source of nitrogen, 4
— , excretion by CI. pastenrianum,

.54 . .
— , inhibition of nitrate reduction,

34

— , — of nitrogen-fixation, 54

— , intermediate in nitrogen- fixa-
tion, 52

— , liberation by deaminases, 10-26

— , oxidation, 32

Anaerobic a-deaminases, 22

Anaerobic metabolism, amino-
acids as H-acceptors, 17

, nitrate as H-acceptor, 39

Anthranilic acid, excretion by
Salm. typhosa, 64

, 3-hydroxy-, 72

, in tryptophan synthesis, 64,

. .71

Antibiotic polypeptides, composi-
tion, 97

Antibiotics, 151

Arginine, as essential amino-acid,26

— , as possible energy source, 27

— , cycle, 69

— , decarboxylase, 27

— , dihydrolase, 26

— , exacting mutants, 69

Asparaginase, 98

Aspartase, 22, 60

Aspartic acid, deamination, 23

, in arginine synthesis, 70

, in nitrogen-fixation, 55

, transamination, 61-3

Aspergillus nidulans, use of mut-
ants, 66, 75

Aspergillus niger, L-amino-acid
oxidase, 11

, synthesis of cysteine, 75

, use of mutants, 66

159

i6o

NITROGEN METABOLISM

Aspergillus parasiticus, peptidases,

124
Athiorhodaceae, 5
Aureomycin, 152, 155
Autotrophs, nitrifiers, 32
— , nutrition, 4
Auxotroph, definition, 69
Azotobacter agilis, a-ketoglutarate,

63

, nitrate reduction, 39

, nitrogen fixation, 47

Azotobacter chroococcum, 47
Azotobacter indicum, 47

B12 and pseudo-Bi2, 137, 149, 151
Bacillus, extracellular peptides, 96
— , nitrate reduction, 39
— , proteases, 120
Bacillus anthracis, peptide cap-
sule, 96

, transforming factors, 134

Bacillus megatherium, peptidases,

Bacillus mycoides, deamination, 1 1
Bacillus subtilis, asparaginase, 98

, gelatinase, 1 20-1

, mutants, 66

, transamination, 61

, tryptophan oxidation, 15

Bacteriophage, synthesis, 140
Bacterium cadaveris, anaerobic

deaminases, 24
Barbituric acid, 136
Biocytin, 24
Biotin, in ammonia assimilation

by yeast, 25
— , in deaminase activation, 24
Brucella abortus, transamination, 63

Calcium, in formation of extra-
cellular proteinases, 120

— , in gelatinase production, 120

— , in nitrification, 33

— , in nitrogen-fixation, 47

Calothrix, nitrogen-fixation, 50

Carbamylglutamic acid, in citrul-
line synthesis, 70

Carbon monoxide, inhibition of
nitrogen-fixation, 52

Carbon source, 4

■ , carbon dioxide as, 4

Carboxypeptidases, 113

Chloramphenicol (Chloromycetin),
152, 155, 156

Chromatinic bodies, 133

CitruUine, from arginine, 26

Citrulline, from ornithine, 69-71

Clostridium, amino-acid decar-
boxylases, 27

— , nitrogen-fixation, 49

— , proteases, 116-20

Clostridium acidi-urici, purine fer-
mentations, 136

Clostridium botulinum, extracellu-
lar proteinases, 119

Clostridium cochlearum, amino-
acid fermentations, 22

Clostridium cylindrosporum, uric
acid fermentation, 136

Clostridium histolyticum, extracel-
lular proteinases, 117, 119

Clostridium kluyveri, acetylation
of amino-acids, 104

Clostridium pasteurianum, isola-
tion, 46

, nitrogen-fixation, 51, 54-5

Clostridium propionicum, amino-
acid fermentations, 21

Clostridium septicum, arginine di-
hydrolase, 26

, proteinases, 117, 119

Clostridium sporogenes, arginine
dihydrolase, 26

• , glutamic dehydrogenase, 1 2

, Stickland reaction, 17

Clostridium tetani, amino-acid fer-
mentations, 22

, and glutamine, 98

Clostridium tetanomorphum, amino-
acid fermentations, 21

Clostridium welchii and glutamine,
98

, collagenase (< toxin), 118

, A toxin, 119

, nitrate reduction, 39

Cobalamins, see B12

Codecarboxylase, see Pyridoxal
phosphate

Coenzyme A, 3, 103-4

Collagenase, 116

Corynebacterium, pyrimidine fer-
mentations, 136

Corynebacterium diphtheriae, pep-
tides for growth, 102

Cystathionine, 74

Cysteine, desulphurase (deami-
nase), 23

— , sulphinic acid, 75

— , -S-sulphonate, 75

— , synthesis, 75

Cytosine, 126

— , 5-methyl-, 126

INDEX

;6i

Deaminases, amino-acids, aerobic,

10-17
— , — , anaerobic, 22-6
— , purines, 136
5-Dehydroquinic acid, 73
Denitrification, economic import-
ance, 43
— , effect of oxygen, 42
— , organisms, 39
— , pathways, 42

Deoxypentose nucleic acids, in
chromatinic bodies,

133
, in transforming factors,

134

■ , in viruses, 140

2-Deoxyribose, 127, 139
Desaturases, see Deaminases, an-
aerobic
Desulphovibrio, nitrogen-fixation,

50
Diamine oxidase, 155
a,e-Diaminopimelic acid, decar-
boxylase, 29

, in lysine synthesis, 75

a,;?-Dihydroxy-iS-ethylbutyricacid,

76.
Dipeptidases, 124
Diplococcus glycinophilus, 20
Drugs, mode of action, 145

Energy sources, 4, 17, 20
Energy transfer in biological sys-
tems, 3
Enrichment cultures, 20, 46
Enzymes, adaptive, 13
■ — , control of formation by genes,

^7 .
Escherichia coli, amino-acid de-
carboxylases, 27

, aspartase, 23

, cysteine desulphurase,

23

, cytidine deaminase, 136

, cytosine deaminase, 136

, deamination of amino-acids,

II

, — of histidine, 22

, dephosphorylation of

nucleotides, 135

, glutathione synthesis, 104

, infection by bacteriophage,

140
, mutants, utilization of

amino-acids in peptides,

100

Escherichia coli, nitratase, 40

, — , synthesis of, 106

, purine interconversion, 138

, purine synthesis, 137

, serine and threonine de-
aminases, 23

, transamination, 60

, transforming factors, 134

, tryptophanase, 15

Extracellular products, anti-
biotics, 96
— ■ — , enzymes, 112, 115

, peptides, 96-7

• , proteinases, 115

Fermentation, amino-acids, 20

— , purines, 136

— , pyrimidines, 136

Folic acid factors, structure, 98,

Folinic acid (citrovorum factor),

147
Formate, in purine synthesis, 137
Formylase, 13
Formylkynurenine, 13
Fusarium lycopersici, 102
Fusel oil, 22

Gelatinase, 116

Glucose, effect on tryptophanase
formation, 15

D-Glutamic acid in extracellular
peptides, 96

L-Glutamic acid, conversion to
ornithine, 71

, — to proline, 70

, dehydrogenase, 12, 54, 60,

64

, in folic acid factors, 98

, in histidine catabolism, 22

, in nitrogen-fixation, 53

, in strepogenin, 102

, in transamination, 61

, racemase, 95

, uptake by cocci, 86

Glutamine, in citrulline synthesis,
70

— , initiation of growth, 96^ 98

— , synthesis, 104

Glutathione, activation of deami-
nases, 24

— , glyoxalase, 99

— , occurrence, 99

— , reductase, 99

— , structure, 99

— , synthesis, 104

1 62

NITROGEN METABOLISM

Glutathione, transpeptidation re-
actions, 99

— , triose phosphate dehydro-
genase, 3, 99

Glycine, and Lb. easel, loi

— , fermentation, 20

— , in purine synthesis, 137

— , oxidation, 11

Gram stain, 132

Growth, and nucleic acid content,
107

Growth conditions, effect on
amino-acid decarboxy-
lases, 28

, deaminase activity, 25

, extracellular proteases,

120

, tryptophanase, 15

Guanine, 126

Haemoglobin, in legume root
nodules, 57

Haemophilus influenzae, X and V
factors, 12

, transforming factors, 134

Haemophilus parainfluenzae, and
putrescine, 29

, glutamic dehydrogenase, 12

Haemophilus pertussis and gluta-
mic acid, 12

Hansenula, differentiation from
Pichia, 43

Hansenula anomala, nitrate reduc-
tion, 39

Heterocaryosis, 68

Histidine, a-deaminase, 21

— , fermentation, 21

— oxidation by Ps. fluorescens, 22
Homoserine, 75

Hydrogen acceptors, i
Hydrogen donors, i
Hydrogenase, and nitrate reduc-
tion, 41

— and nitrogen-fixation, 5 1

— and Stickland Reaction, 19
p-Hydroxybenzoic acid, 73
Hydroxylamine, in denitrification,

— , in nitrification, 37
— , in nitrogen-fixation, 55, 57
Hyponitrous acid, in denitrifica-
tion, 42

Indole, from tryptophan, 15

• — in tryptophan synthesis, 64, 71

Indoleacrylic acid, 64

Inhibitors, metabolite analogues,

64
Intracellular proteases, 114
Ion transport, 89
Isoleucine and B. anthracis^ 65
— , synthesis, 76
Isotopes and study of amino-acid

synthesis, 77
Isotopic nitrogen and pathways of

nitrogen-fixation, 53

a-Ketoglutaric acid, synthesis, 63
Kynureninase, 14
Kynurenine, in tryptophan de-
gradation, 13
Kynureninic acid, 13

Lactobacillus, amino-acid decar-
boxylases, 27

— , peptidases, 124

Lactobacillus arabinosus and gluta-
mine, 98

and quinolinic acid, 72

, transaminases, 62

, utilization of peptides, 100

Lactobacillus bifidus, serine syn-
thesis, 149

Lactobacillus casei, and aspartase,

, folic acid requirement, 98

, purine interconversion, 138

, strepogenin, 102

— — , utilization of peptides, loi

Lactobacillus delbriickii, utiliza-
tion of peptides, loi

Lactobacillus helveticus, nucleo-
sidases, 135

Lactobacillus lactis and Bic, 149

Lactobacillus pentosus, nucleosi-
dases, 136

Leghaemoglobin, see Haemoglo-
bin

Leucine and B. anthracis, 65

Leuconostoc mesenteroides, and as-
paragine, 98

, and glutamine, 98

, peptidases, 124

, serine synthesis, 148

, utilization of peptides, 100

Lycomarasmin, 102

Lysine, synthesis, 75

— , uptake by cocci, 83

— , — by yeast, 86

Magnesium and extracellular pro-
teases, 120

INDEX

163

iVIanganese, activation of pepti-
dases, 123

Metabolite analogues, 64

Metals, activation of proteases,
120, 123

Methionine, in purine svnthesis,
137-8

— , sulphonamide antagonist, 147

— , synthesis, 74, 149

Micrococcus aerogenes, purine fer-
mentations, 136

Micrococcus anaerobius, glycine
fermentation, 20

Micrococcus denitrificans, nitrate
reduction, 39

Micrococcus lactilyticus, purine
fermentations, 136

Micrococcus variabilis, glycine fer-
mentations, 20

Molybdenum and nitrogen-fixa-
tion, 50

Mutants, methods of isolation,
66-7

— , production, 66

— , spontaneous, 66

— , use of penicillin, 68

Mycobacterium, pyrimidine fer-
mentations, 136

— , streptomycin and diamine
oxidase, 155

Mycobacterium tuberculosis, pepti-
dases, 124

Neiirospora, adenosine deaminase,
136

— , arginine synthesis, 69

— , nitratase, 40

— , reproductive cycles, 66

— , transaminases, 61

— , use of mutants, 66

Neiirospora crassa, D-amino-acid
^ oxidase, 11

r , L-amino-acid oxidase, 1 1

, L-glutamic acid dehydro-
genase, 12

, nitrate reduction, 39

Nicotinic acid, relation with trj'p-
tophan, 72

Nitramide, in denitrification, 41

Nitratase, 40

Nitrate and nitrogen-fixation, 54

• — from nitrite, 32

■ — reduction, 39

Nitric oxide in denitrification, 42

Nitrification, effect of organic
compounds, 34

Nitrification, effect of Chloro-
mycetin, 38

— , — of potassium chlorate, 38

— , energy relationships, 39

— , hydroxylamine in, 37

— , in soil, 36

— , isolation of organisms, 33

— , soil percolation apparatus, 35

Nitrite, oxidation to nitrate, 32

Nitrobacter zvinogradsky , 33

Nitrogen cycle, 6

Nitrogen-fixation by blue-green
algae, 50, 56

by Clostridium, 49

by Desulphovibrio, 50

by leguminous plants, 45

by photosynthetic organ-
isms, 50

detection of, 47

effect of pCO, 52

of pHa, 51

of pNa, 51

of trace elements, 50-1

isolation of organisms, 46

pathways, 56

Nitrogen sources, 4

Nitrosomonas europoea, 33

Nitrous oxide, in denitrification,
42

Nitroxyl, 42

Nocardia, pyrimidine fermenta-
tion, 136

Nodules, nitrogen-fixation by, 50

Nostoc and nitrogen-fixation, 50

Nucleic acids, chromatinic bodies,
133

, components, estimation of,

129

, composition, 127

, Gram stain, 132

, hydrolysis of, chemical, 130

, enzymic, 134

, microbial, 131

, protein synthesis, 107

, structure, 127

, transforming factors, 134

Nuclecproteins, 127

Nucleosidases, 135

— , in nucleotide synthesis, 140

Nucleosides, structure, 126

Nucleotides, structure, 126-8

Nutrition, 3

Ophiostoma, mutants, 66, 75, 137
Orotic acid, 139
Oxido-reduction reactions, i

164

NITROGEN METABOLISM

Pantothenic acid in Co. A, 150
Penicillin, 151

— and amino-acid uptake, 151
Penicillium notatum, L-amino acid

oxidase, 11

, arginine synthesis, 70

Pepsin, 113

Peptidases, activation of, 123

— , endo-, 113

— , exo-, 113

— , intracellular, 114

— , microbial, 123

Peptide bond synthesis, 103

Peptides, as growth factors, 10 1

— , microbial, composition, 96

— , source of essential amino
acids, 100-3

— . synthesis, 103

Phenylalanine and tryptophanase
formation, 16

Phosphate compounds, energy-
rich, 2

— of nucleic acids and protein

synthesis, 109

Photosynthetic organisms, nitro-
gen-fixation, 50

Phytotnonas tumifaciens, pepti-
dases, 124

Pichia, differentiation from Han-
senula, 43

Plasmin, 122

Plasminogen, 122

Pneumococcus transforming fac-
tors, 134

Polyol phosphates in Staph, aureus,

Propionibactena, peptidases, 124

Protein synthesis, 103

Proteins, decomposition, ii2

— , microbial, 95

— , synthesis, 106

Proteolytic enzymes of bacteria,

115

of Clostridia, 116

of mammals, 113

of staphylococci, 122

of streptococci, 122

Proteus, amino-acid decarboxy-
lases, 27

— , proteases, 120

— , tryptophanase, 15

Proteus morganii, glutamine de-
composition, 98

Proteus vulgaris, L-amino-acid oxi-
dase, II

, anaerobic deaminases, 23

Proteus vulgaris, asparaginase, 98

, aspartase, 23

, cysteine desulphurase, 23

, growth on proteins, 122

, nitrate reduction, 40

, nutrition, 4

, transforming factors, 134

, transpeptidation reaction,

100

Pseudomonas, adaptation, 13

— , proteases, 120

— , tryptophan oxidation, 13

Pseudomonas aeruginosa (Ps. pyo~
cyanea), asparaginase,
98

, peptidases, 124

, serine and threonine

deaminase, 23

, transaminases, 61

Pseudomonas denitrificans, nitrate
reduction, 39

Pseudomonas fluorescens, aspar-
tase, 23

, a-ketoglutarate, 63

, nitrate reduction, 39

, peptidases, 124

, transaminases, 61

Pseudomonas stutzeri, nitrate re-
duction, 39, 41

Purines, fermentation, 136

— , oxidation, 136

— , synthesis, 137

Putrefaction, 112

Putrescine, as growth factor, 29

Pyrimidines, fermentation, 136

— , oxidation, 136

— , synthesis, 138

Pyridoxal phosphate in amino-
acid decarboxylases, 28

in cysteine desulphurase, 25

in racemases, 95

in D and l serine deami-
nases, 25

in transaminases, 61

in ti-yptophan synthesis, 72

in tryptophanase, 16

Pyridoxamine phosphate in trans-
amination, 62

Pyrocatechase in Pseudomonas, 14

Quinic acid, 5-dehydro-, 73
Quinolinic acid, 72

Reduction of amino-acids, 17

— of nitrate, 39

— of nitrite, 40

INDEX 165

Rhizobiiwi, cross-inoculation

groups, 46

— , isolation, 46

Rhodomicrohium and nitrogen-
fixation, 50

Rhodopseudomonas and nitrogen-
fixation, 50

Rhodospirillum and nitrogen-fixa-
tion, 50

Ribonuclease, 134

Ribose, 127, 140

Saccharomyces cerevisiae, biotin
and ammonia assimila-
tion, 25
, glutamic acid dehydroge-
nase, 12

, a-ketoglutarate, 63

, peptidases, 124

Salmonella paratyphi, histidine de-
composition, 22
Salmonella typhosa, nutrition, 4

, tryptophan synthesis, 64

Serine, fermentation by CI. pro-

pionicum, 21
— , in tryptophan synthesis, 64, 72
— , synthesis, 149
Serine deaminases, 23-5
Serratia marcescens, aspartase, 23

, nitrate reduction, 40

, peptidases, 124

Sewage purification, 32
Shigella paradysenteriae, histidine
decomposition, 22

, transforming factors, 134

Shikimic acid, 73

, 5-dehydro-, 73

Simultaneous adaptation, 13
Staphylococcus, serine and threo-
nine deaminase, 23
Staphylococcus aureus and penicil-
lin, 151

arginine dihydrolase, 26

glutamine synthesis, 104

internal free amino-acids, 82

, polyol phosphates, 133

, protein synthesis, 107

Stickland reaction, 17
Strepogenin, 102

Streptococcus, amino-acid decar-
boxylases, 27
— , glutamine decomposition, 98
— , Gp. A, proteinase, 122
Streptococcus faecalis, arginine di-
hydrolase, 26
, biotin and aspartic acid, 24

Streptococcus faecalis, folic acid
requirement, 98

, transaminases, 61

, uptake of amino-acids, 82

, utilization of peptides, loi

Streptococcus haemolytiais and
glutamine, 98

nucleases, 135

streptokinase, 122

Streptococcus lactis, asparagine as

growth factor, 98
Streptokinase, 122
Streptomycin, 152, 155
Sulphonamides, mode of action, 1 46
Synthesis, alanine, 61
— , amino-acids, 60
— , arginine, 69
— , aspartic acid, 60-1
— , bacteriophage, 140
— , cysteine, 73
— , deoxyribose, 139
— , glutamic acid, 60-1
— , isoleucine, 76
— , leucine, 65
— , lysine, 75
— , methionine, 73
— , nucleosides, 140
— , nucleotides, 140
— , ornithine, 71
— , phenylalanine, 73
— , proline, 70
— , purines, 137
— , pyrimidines, 138
— , ribose, 140
— , serine, 149
— , threonine, 75
— , tryptophan, 64, 71
— , tyrosine, 72
— , valine, 76
Syntrophism, 69

Tetrahymena gelei and purines, 138

Thiohacillus denitrificans, nitrate
reduction, 40

Thiorhodaceae, 5

Thiosulphate in cysteine syn-
thesis, 75

Threonine, 75

— , fermentation, 21

Thymine, 126-7

Tonilopsis utilis, isotopes and
amino-acid synthesis, 77

Transaminases, 60

— , bacteria, 6i

— , Neurospora, 61

— , yeasts, 6i

1 66

NITROGEN METABOLISM

Transforming factors, 134
Transpeptidation, 99, 104
Triose phosphate dehydrogenase,

3» 99
Tryptophan and nicotinic acid, 71

— and Salm. typhosa, 64

— cycle, 72

— decomposition by Pseudomo-

nas spp., 13

, aromatic pathway, 14

, quinohne pathway, 14

— peroxidase in Pseudomonas, 13

— synthesis, 64, 71

, inhibitors, 64

Tryptophanase, 15
— , mechanism, 16
Tyrosine, decarboxylase, 27

— , effect on tryptophanase forma-
tion, 16

Ultraviolet light, absorption by
purines and pyrimidines,
129

, induction of mutation, 66

Ultraviolet light, inhibition of
adaptation, 107

, use in photographing chro-

matograms, 131
Uracil, 126

Ureidosuccinic acid, 139
Uridine diphosphate glucose, 127
Uridine-5-pyrophosphate pep-
tides, 15s
Urocanic acid, 21

Valine and Bacillus anthracis, 65
Vibrio, tryptophanase, 15

Yeast, alcohols from amino-acids,

22
— , aspartase, 23
— , glutamine synthesis, 104
— , nucleosidase, 136
— , purine synthesis, 137
— , transaminases, 61

Zinc, activation of serine deami-
nase, 25