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C O M P R E H E N S I VE B I O C H E M I S T RY
ADVISORY BOARD
SIR RUDOLPH A. PETERS, M.C., M.D., D.Sc, F.R.S.
Emeritus Professor of Biochemistry, Oxford ; Department of Biochemistry, Cambridge
Chairman
CF. CORI, M.D., D.Sc.
Professor of Biochemistry, Washington University, School of Medicine, St. Louis, Mo.
J.N. DAVIDSON, D.Sc, M.D., F.R.S.
Professor of Biochemistry, The University of Glasgow, Scotland
E. F. GALE, D.Sc, F.R.S.
Professor of Chemical Microbiology, University of Cambridge
A. BAIRD HASTINGS, B.Sc, PH.D., D.SC.
Director of Biochemistry Division,
Scripps Clinic and Research Foundation, La Jolla, Calif.
E. LEDERER, PH.D., D.SC.
Professor of Biochemistry, Faculty of Science, University of Paris
F. LYNEN
Max Planck Institute for Cell Chemistry, Munich
R. NICOLAYSEN, M.D.
Professor of Nutrition Research, University of Oslo
S. OCHOA, B.A., M.D., HON.LL.D., HON.D.SC.
Professor of Biochemistry, New York University School of Medicine, New York, N.Y.
J. ROCHE, D.Sc
Professor of General and Comparative Biochemistry, Collège de France, Paris
KENNETH V. THIMANN
Professor of Biology, Harvard University, Cambridge, Mass.
A. W. K. TISELIUS, D.Sc, FOR.F.R.S.
Professor of Biochemistry, Institute of Biochemistry, Uppsala, Sweden
H. G. K. WESTENBRINK (deceased)
Professor of Physiological Chemistry, State University, Utrecht, The Netherlands
F. G. YOUNG, M.A., D.Sc, F.R.S.
Professor of Biochemistry, University of Cambridge
C O M P R E H E N S I VE
B I O C H E M I S T RY
SECTION I (VOLUMES 1-4)
PHYSICO-CHEMICAL AND ORGANIC ASPECTS
OF BIOCHEMISTRY
SECTION II (VOLUMES 5-11)
CHEMISTRY OF BIOLOGICAL COMPOUNDS
SECTION III (VOLUMES I2-l6)
BIOCHEMICAL REACTION MECHANISMS
SECTION IV (VOLUMES I7-2l)
METABOLISM
22-2j)
SECTION V (VOLUMES
CHEMICAL BIOLOGY
GENERAL INDEX (VOLUME 28)
C O M P R E H E N S I VE
B I O C H E M I S T RY
EDITED BY
M A R C EL F L O R K IN
Professor of Biochemistry, University of Liège (Belgium)
AND
E L M ER H. S T O TZ
Professor of Biochemistry, University of Rochester, School of Medicine
and Dentistry, Rochester, N.Y. (U.S.A.)
V O L U ME 16
HYDROLYTIC REACTIONS;
COBAMIDE AND BIOTIN COENZYMES
E L S E V I ER P U B L I S H I NG C O M P A NY
AMSTERDAM·LONDON·NEW YORK
1965
CONTRIBUTORS TO THIS VOLUME
H.A. BARKER, PH.D.
Professor of Biochemistry, Department of Biochemistry, University of California,
Berkeley, Calif. 95054 (U.S.A.)
LEON W. CUNNINGHAM, PH.D.
Professor of Biochemistry, Department of Biochemistry, School of Medicine, Station 17,
Vanderbilt University, Nashville, Tenn. 37203 (U.S.A.)
H. S. JANSZ, PH.D.
Laboratory of Physiological Chemistry, University of Leiden (The Netherlands)
YOSHITO KAZIRO, M.D., PH.D.
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Bunkyo-ku,
Tokyo (Japan)
ROBERT K. MORTON*, PH.D.
Professor of Biochemistry, Department of Agricultural Chemistry, Waite Agricultural
Research Institute and Department of Biochemistry, University of Adelaide (Australia)
SEVERO OCHOA, M.D.
Professor of Biochemistry, Department of Biochemistry, New York University School
of Medicine, 550 First Avenue, New York, N.Y. 10016 (U.S.A.)
R. A. OOSTERBAAN, PH.D.
Medical-Biological Laboratory of the National Defense Research Council—TNO,
Rijswijk (Z.H.), (The Netherlands)
ALAN PETERKOFSKY, PH.D.
Laboratory of Biochemistry, National Institute of Dental Research, National Institutes
of Health, Bethesda, Md. 20014 (U.S.A.)
HERBERT WEISSBACH, PH.D.
Laboratory of Clinical Biochemistry, National Heart Institute, National Institutes of
Health, Bethesda, Md. 20014 (U.S.A.)
* deceased.
GENERAL PREFACE
The Editors are keenly aware that the literature of Biochemistry is already
very large, in fact so widespread that it is increasingly difficult to assemble
the most pertinent material in a given area. Beyond the ordinary textbook
the subject matter of the rapidly expanding knowledge of biochemistry is
spread among innumerable journals, monographs, and series of reviews.
The Editors believe that there is a real place for an advanced treatise in
biochemistry which assembles the principal areas of the subject in a single
set of books.
It would be ideal if an individual or small group of biochemists could
produce such an advanced treatise, and within the time to keep reasonably
abreast of rapid advances, but this is at least difficult if not impossible.
Instead, the Editors with the advice of the Advisory Board, have assembled
what they consider the best possible sequence of chapters written by
competent authors; they must take the responsibility for inevitable gaps of
subject matter and duplication which may result from this procedure.
Most evident to the modern biochemist, apart from the body of knowledge
of the chemistry and metabolism of biological substances, is the extent to
which he must draw from recent concepts of physical and organic chemistry,
and in turn project into the vast field of biology. Thus in the organization
of Comprehensive Biochemistry, the middle three sections, Chemistry of
Biological Compounds, Biochemical Reaction Mechanisms, and Metabolism
may be considered classical biochemistry, while the first and last sections
provide selected material on the origins and projections of the subject.
It is hoped that sub-division of the sections into bound volumes will not
only be convenient, but will find favour among students concerned with
specialized areas, and will permit easier future revisions of the individual
volumes. Toward the latter end particularly, the Editors will welcome all
comments in their effort to produce a useful and efficient source of biochem-
ical knowledge.
Liège/Rochester M. FLORKIN
E. H. STOTZ
PREFACE TO SECTION III
(VOLUMES 12-16)
Following Section II of Comprehensive Biochemistry on the Chemistry
of Biological Compounds, and preceding sections on Metabolism and Chemical
Biology, Section III is devoted primarily to Enzymes. Recognizing the
encyclopedic nature of any effort to provide even a minimal treatment
of all known enzymes, the Editors have chosen instead to select examples
from modern enzymology in which advances in reaction mechanisms have
been made. Certainly a well-established biochemical reaction mechanism
is the carrier function of coenzymes which serve as the prosthetic groups of
enzymes, and Section III has a primary purpose of providing treatment of
both the chemistry and function of the coenzymes. Other chapters, however,
treat thermodynamic and kinetic aspects of enzyme catalysis, hydrolytic
enzymes displaying "active center" characteristics, and chelation and
stereochemical considerations in enzyme catalysis. A considerable portion
of the Section deals with biological oxidation mechanisms. Finally, Section
III would seem incomplete without inclusion of the recommendations of the
Enzyme Commission of the International Union of Biochemistry and the
classified list of Enzymes.
Liège/Rochester M. FLORKIN
Ε. H. STOTZ
Chapter I
Cholinesterases, Esterases and Lipases
R. A. OOSTERBAAN
Medical-Biological Laboratory of the National Defense Research Council—
TNO, Rijswijk (Z.H.), (The Netherlands)
AND
H. S. JANSZ
Laboratory of Physiological Chemistry University
}
of Leiden (The Netherlands)
1. Introduction
The enzymes which will be discussed in this Chapter have in common that
they catalyze the hydrolysis of carboxyl esters. In recent years evidence has
accumulated indicating that enzymatic carboxyl ester cleavage is, in many
cases, governed by a mechanism which is basically similar for different
enzymes. The evidence has been derived from studies on the kinetics of the
interaction of these enzymes with substrates and inhibitors and specifically
from chemical analysis of the active site. The similarity in mechanism may
be due to the presence of a common or at least related active site in different
carboxyl esterases. In Une with the general objective of this series of papers
on biochemical reaction mechanisms an attempt is made to present infor-
mation on enzymes, their substrates and inhibitors which contributes to an
understanding of the molecular basis of the general mechanism of enzymatic
ester hydrolysis. Also included are data which are relevant to an under-
standing of the molecular basis of substrate specificities of individual
enzymes. Evidently, a discussion on a molecular basis must be limited to
those enzymes that have been purified and characterized in detail. Conse-
quently, the discussion will be confined to the few representatives within the
groups of cholinesterases, lipases and esterases that have been extensively
References p. 47
[I]
2 CHOLINESTERASES, ESTERASES, LIPASES I
purified and characterized. For a detailed account of the occurrence and
properties of the many other carboxyl esterases and for alternative aspects
outside the scope of the present paper, the reader is referred to reviews
1-3
available in the literature .
2. Classification of carboxyl esterases
Many enzymes that hydrolyze carboxyl esters exhibit a very broad substrate
specificity. Consequently, a classification based on their substrate specificity
will be of limited value. However, the carboxyl esterases may be designated
as A-, B- or C-esterases on the basis of their reaction with organophosphates
(e.g. DFP). Esterases of the A-type are not inhibited by organophosphates
^OCH(CH )
32
F—p==o
OCH(CH)
32
(1)
Diisopropyl phosphorofluoridate (DFP)
but they hydrolyze these compounds. B-esterases are sensitive to inhibition
by organophosphates in low concentrations. The C-esterases do not hydrolyze
the organophosphates nor are they inhibited by these compounds. Different
4 5
A- and B-types of carboxyl esterases have been found in plasma » . The
67
C-type of carboxyl esterase occurs in hog kidney ' . The separation of an
A-type carboxyl esterase (Α') from two B-type esterases (B' and B") by zone
electrophoresis of horse plasma is shown in Fig. i.
The A' enzyme rapidly hydrolyzes phenyl acetate and is not inhibited by
organophosphates. Aliphatic carboxyl esters are poor substrates for this
enzyme. The B'- and Β "-esterases are both inhibited by organophosphates
in low concentrations. In contrast to the Β'-esterase, the Β "-esterase is
inhibited by physostigmine (eserine) in low concentrations. The sensitivity
to inhibition by physostigmine may be used as a further method for distin-
guishing different types of B-esterases. The physostigmine-sensitive enzymes
hydrolyze choline esters (but also aromatic esters) ; they are usually des-
ignated as cholinesterases. The Β'-esterase hydrolyzes aliphatic as well as
aromatic carboxyl esters. Low physostigmine-sensitive esterases of this type
attacking short-chain fatty acid esters are usually named ali-esterases. The
term lipase is used for enzymes that hydrolyze long-chain fatty acid esters,
e.g. fats. The lipases, however, also hydrolyze short-chain fatty acid esters.
8
It has been suggested by Desnuelle and others to use the term lipase for
enzymes which act on undissolved substrates. The term ali-esterase would
refer to enzymes acting on substrates in solution. This distinction becomes
2 CLASSIFICATION OF CARBOXYL ESTERASES 3
Fraction number
Fig. ι. Distribution of esterase activity and total' protein (—· — ·—) of horse plasma
after column electrophoresis. Esterase activities against phenyl acetate ( • · · · •),
5
phenyl butyrate ( • •) and butyrylcholine (—ο—Ο—). (From Augustinsson ).
vague however, since the carboxyl esterase from wheat germ hydrolyzes
both undissolved and dissolved substrates.
The differences in mechanism of action between Α-, B- and C-types of
esterases may well be of a qualitative rather than of a quantitative character.
It is known that the inhibition of B-type esterases by organophosphates in-
volves a phosphorylation of the active centre of these enzymes. The rate of
inhibition, however, is greatly different for individual esterases of the B-
5
type. Butyrylcholinesterase reacts 5 · io times faster with tetra-ethyl pyro-
phosphate than does trypsin. The difference between a B- and C-type
esterase may then be that the latter reacts with organophosphates at an
extremely slow rate. Likewise, the rate of dephosphorylation may differ
considerably for different types of B-esterases inhibited by organophos-
9
phates .
An A-type esterase may hydrolyze organophosphates because it is rapidly
phosphorylated by organophosphates like some B-esterases but unlike the
latter, the phosphorylated Α-esterase is rapidly dephosphorylated. In this
10
way Van Asperen and Oppenoorth explained the observed development of
resistance of house flies against organophosphates. The resistance was
presumed to be due to a single gene mutation which causes the synthesis of
an A-type of esterase instead of the usual B-type which is present in the
wild type.
It should be noted that another group of enzymes from tissues (DFPase,
References p. 47
