Chemical Pathways of Metabolism

385 Pages·1954·22.699 MB·English

Chemical Pathways
of
Metabolism
EDITED BY
DAVID M. GREENBERG
Department of Physiological Chemistry
School of Medicine
University of California
Berkeley, California
VOLUME II
1954
ACADEMIC PRESS INC., PUBLISHERS
NEW YORK, N. Y.
Copyright 1954, by
ACADEMIC PRESS INC.
125 EAST 23RD STREET
NEW YORK 10, N.Y.
All Rights Reserved
No part of this book may be reproduced in any
form, by photostat, microfilm, or by any other
means, without written permission from the
publishers.
Library of Congress Catalog Card Number: 54-7613
PRINTED IN THE UNITED STATES OF AMERICA
CONTRIBUTORS TO VOLUME II
H. BORSOOK, Kerckhoff Laboratories of Biology, California Institute of
Technology, Pasadena, California
P. P. COHEN, Department of Physiological Chemistry, University of
Wisconsin, Madison, Wisconsin
S. GRANICK, Rockefeller Institute for Medical Research, New York, New
York
D. M. GREENBERG, Department of Physiological Chemistry, School of
Medicine, University of California, Berkeley, California
LEON A. HEPPEL, National Institutes of Health, U. S. Public Health
Service, Bethesda, Maryland
MARTIN P. SCHULMAN, Department of Biochemistry, State University of
New York Medical School, Syracuse, New York
V
LIST OF ABBREVIATIONS AND SYMBOLS
AMP adenosine-5-phosphate
ADP adenosine diphosphate
ATP adenosine triphosphate
DPN , DPN+, DPN diphosphopyridine nucleotide (cozy-
OI
mase, coenzyme I)
DPN , DPNH reduced form of above
red
TPN , TPN+ TPN , TPNH triphosphopyridine nucleotide (coen­
OI red
zyme II)
FAD flavin adenine dinucleotide
CoA, CoASH coenzyme A
RSH sulfhydryl compounds
SH S
/ /I
L ,L lipoic acid, thioctic acid
\ \
SH S
Kcal. kilocalories
Kj. kilojoules
metabolic quotients expressed in μΐ.
'ΐθ2? Vacetatej ClC.
metabolite/mg. dry weight/hr.
AF increment of free energy
AF° standard free energy change
RNA ribose nucleic acid
PNA pentose nucleic acid, desoxyribose nu­
cleic acid
Pi inorganic phosphate
PPi inorganic pyrophosphate
PPPi inorganic triphosphate
Contents of Volume I
1. Free Energy and Metabolism by ARTHUR B. PARDEE
2. Enzymes in Metabolic Sequences by DAVID E. GREEN
3. Glycolysis by P. K. STUMPF
4. The Tricarboxylic Acid Cycle by H. A. KREBS
5. Other Pathways of Carbohydrate Metabolism by SEYMOUR S. COHEN
6. Biosynthesis of Complex Saccharides by W. Z. HASSID
7. Fat Metabolism and Acetoacetate Formation by I. L. CHAIKOFF and G. W.
BROWN, JR.
8. Sterol and Steroid Metabolism by D. K. FUKUSHIMA and ROBERT S. ROSENFELD
Author Index
Subject Index
CHAPTER 9
Nitrogen Metabolism of Amino Acids
P. P. COHEN
Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin
Page
I. Scope 1
II. Deamination 1
1. Oxidative 1
a. L-Amino Acid Oxidases (Dehydrogenases) 4
b. D-Amino Acid Oxidases (Dehydrogenases) 11
c. Specific Amino Acid Oxidases (Dehydrogenases) 14
d. Amine Oxidases (Dehydrogenases) 16
2. Nonoxidative Deamination 22
a. Amino Acid Deaminases 22
b. Cysteine and Homocysteine Desulfhydrases 24
c. Other Nonoxidative Deaminases 25
III. Deamidation 26
a. Amino Acid Amidases 26
IV. Transamination 29
V. Amino Acid Racemases 36
VI. Urea Synthesis 36
VII. Summary Remarks 43
Addendum 45
I. SCOPE
This chapter is concerned primarily with the metabolic reactions in­
volved in transforming or transferring the amino, amine, and amide
nitrogen moiety of amino acids, amino acid amides, and amines. Although
reference may be made to ill-defined enzyme systems, emphasis will be
placed on those systems which have been at least partially purified and
reasonably well studied.
Recent reviews of certain aspects of this subject have appeared.1>2'3
II. DEAMINATION
1. OXIDATIVE
A large number of enzyme systems have been recorded in the literature
in support of the concept that amino acids as a group are oxidatively
1 Krebs, H. A., Enzymes 2 (pt. 1), 499-535 (1951).
2 Tarver, H., In D. M. Greenberg, Amino Acids and Proteins, pp. 769-908, Charles
C Thomas, Springfield, Illinois, 1951.
3 Tarver, H., Ann. Rev. Biochem. 21, 301 (1952).
1
2 P. P. COHEN
deaminated in accordance with the following general over-all reaction:
RCHCOOH + O - -> RCCOOH + NH (1)
:i
I II
NH, O
In a general way these enzyme systems may be classified as follows:
1. L-Amino acid oxidases (or dehydrogenases).
2. D-Amino acid oxidases (or dehydrogenases).
3. Specific amino acid oxidases (or dehydrogenases).
This classification indicates that the L- and D-amino acid oxidases have
broad substrate specificities, whereas the other enzymes have a narrower
specificity. For the purpose of simplicity the above classification will
be used. The amino acid oxidase systems may be subclassified according
to the nature of the hydrogen acceptor under two categories, aerobic
and anaerobic. The former have been more commonly referred to as
amino acid oxidases, whereas the latter are frequently referred to as
dehydrogenases.
The term oxidase is usually considered to represent a system in which
oxygen is an obligatory hydrogen acceptor. The term dehydrogenase is
used for that enzymatic component of an oxidative reaction system which
is concerned with the activation of the substrate and its dehydrogenation.
Thus, the distinction between the succinoxidase system and succinic
dehydrogenase is clearly recognized under this terminology. In the case
of the amino acid oxidative enzymes, the distinction is somewhat more
difficult to establish, owing chiefly to the fact that most of the measure­
ments of activity have been based on oxygen consumption and thus it is
not certain in most instances that the rate-limiting reaction is the dehy­
drogenase step. It is clear from the formulation of the mechanism of the
oxidation of amino acids and amines that the primary reaction is that of a
dehydrogenation and that the ultimate fate of the hydrogen (or electrons)
is determined either by the autoxidizability of the primary acceptor,
i.e., coenzyme, or by its participation in the chain of hydrogen or
electron transport systems of the cell. The term oxidase will be retained
in referring to most of the amino acid oxidizing enzyme systems be­
cause of precedent and for the reasons given above. The term dehydro­
genase will be included parenthetically in those instances in which in
the author's opinion the term is preferable to the more commonly used
term, oxidase. It is beyond the scope of this discussion to reclassify and
rename the many enzymes mentioned. It should be clear to the reader,
however, that there is a great need for a more systematic basis for
nomenclature.
NITROGEN METABOLISM OF ΑΜΙΝΟ ACIDS 3
Reaction 1 should be considered as a two-step reaction as follows:
RCH—COOH ;=± RC—COOH + 2H' (2)
NHRi NRi
RC—COOH ;=L± RCOCOOH + NH^ (3)
|| -H2O
NR!
Thus, the primary oxidative step is a dehydrogenation (and thus the term
dehydrogenase is preferable) to form an imino acid which in turn is non-
enzymatically hydrolyzed to form the keto acid and ammonia or a deriva­
tive thereof. In the formulation of reactions 2 and 3, Ri is intended to
represent either a hydrogen atom or a substituent, such as an alkyl group.
If Ri is a hydrogen atom, ammonia is formed; if Ri is an alkyl group, an
alkyl amine is formed.
The aerobic oxidases, as far as they have been investigated, are all
flavoproteins, and the hydrogen acceptor for this group is molecular
oxygen. The nature of the oxidative steps following reaction 2 may be
represented as follows:
R R H
1 N
^ C=0
1 1 (4)
X^
II H II
0 0
seaR R
H
C=0
+ H 0 (5)
. /NH 2 2
vc C
H II
II 0
0
The net effect of reactions 4 and 5 is:
2H +O2-+ H 0 (6)
2 2
Thus, the aerobic oxidases form hydrogen peroxide. In the absence of
catalase, the peroxide formed may react with the keto acid as follows:
RCOCOOH + H 0 -> RCOOH + C0 + H 0 (7)
2 2 2 2
The over-all formulation of amino acid oxidation by an aerobic oxidase
in the absence of catalase is as follows:
4 P. P. COHEN
RCH—COOH + 0 -+ RCOOH + NHRi + C0 (8)
2 2 2
NHR!
In the presence of catalase which decomposes the hydrogen peroxide,
the over-all reaction is:
RCH—COOH + y0 -> RCOCOOH + NH^ (9)
2 2
I
NHRi
Keilin and Hartree4 demonstrated the coupling of D-amino acid oxidase
with ethyl alcohol in the presence of catalase and were able to show that
in the presence of catalase ethyl alcohol was oxidized in preference to the
α-keto acid to form acetaldehyde as follows:
catalase
CHCHOH + H 0 > CH3CHO + 2H0 (10)
3 2 2 2 2
The total coupled system is thus formulated as follows:
RCH—COOH + 0 + CHCHOH -> RCOCOOH + NH2R1 + CH3CHO + H0
2 3 2 2
NHR!
(11)
and exhibits the theoretical oxygen uptake without destruction of the
α-keto acid.
The anaerobic oxidases (dehydrogenases) represent enzymes which
have as coenzymes nonautoxidizable hydrogen acceptors and thus are
linked with the cytochrome system. These systems do not produce hydro­
gen peroxide.
a. h-Amino Acid Oxidases (Dehydrogenases)
Three L-amino acid oxidases have been partially purified and studied,
all from such widely different sources as snake venom, rat kidney, and
molds.
(1) Snake Venom or Ophio L-Amino Acid Oxidase (Dehydrogenase).
Zeller and Maritz (see5) described an enzyme originally discovered in snake
venoms and later shown to be present in various tissues of both venomous
and nonvenomous snakes, which they termed ophio L-amino acid oxidase.
4 Keilin, D., and Hartree, E. F., Proc. Roy. Soc. (London) 119B, 114 (1936).
NITROGEN METABOLISM OF AMINO ACIDS 5
The enzyme is widely distributed in a large number of snake venoms. 5
The L-amino acid oxidase from moccasin venom has been highly purified
and shown to be a homogeneous protein with a molecular weight of 62,000.6
It has a turnover number of 3100, a Q of 68,400, and a pH optimum of
0i
7-7.5, with a sharp decline in activity on either side.6 The prosthetic group
has been shown to be FAD, which is present in a concentration of 1 mole
per mole of protein.7
(a) Specificity. Early specificity studies have been reviewed by Zeller 5
and more recently carried out by Bender and Krebs. 8 In Table I are listed
the reactivities of different amino acids with four different amino acid
oxidases, as reported by Bender and Krebs. 8 In general, ophio-L-amino
acid oxidases, although showing variation in substrate specificity from
one species to another, are highly specific for L-amino acids. Zeller5 has
summarized the specificity requirements as follows: "The substrate must
possess a free carboxyl group, an unsubstituted α-amino group and an
organic radical. A second amino or carboxyl group inhibits a substance
otherwise suitable as a substrate for the enzyme."
A study of twenty-two amino acid analogs using cottonmouth snake
venom9 revealed that all were oxidized with the exception of two which
had a tertiary α-carbon. A variety of ß-arylalanines was found to be
oxidized at rates comparable to that of the corresponding naturally
occurring amino acids, supporting the generalization of Zeller5 that the
ß-group exerted relatively little influence on the substrate activity of
different amino acids.
(b) Mechanism of Action. With the clear demonstration by Singer and
Kearney7 that FAD is the coenzyme of ophio-L-amino acid oxidase, the
oxidative steps are those represented by reactions 2, 4, and 5. In the
absence of catalase, the over-all reaction is that shown by reaction 8; in
the presence of catalase, it is that shown by reaction 9. The reaction pro­
ceeds more rapidly in pure oxygen than in air. 6
On the basis of pH activity data and reversible inactivation by phos­
phate and other ions, Kearney and Singer 10-12 have suggested that one of
the active groups of the enzyme is an ionizable imidazole.
5 Zeller, E. A., Advances in Enzymol. 8, 459 (1948).
6 Singer, T. P., and Kearney, E. B., Arch. Biochem. 29, 190 (1950).
7 Singer, T. P., and Kearney, E. B., Arch. Biochem. 27, 348 (1950).
8 Bender, A. E., and Krebs, H. A., Biochem. J. (London) 46, 210 (1950).
9 Frieden, K, Hsu, L. T., and Dittmer, K., J. Biol. Chem. 192, 425 (1951).
10 Kearney, E. B., and Singer, T. P., Arch. Biochem. 33, 377 (1951).
11 Kearney, E. B., and Singer, T. P., Arch. Biochem. 33, 397 (1951).
12 Kearney, E. B., and Singer, T. P., Arch. Biochem. 33, 414 (1951).