Table Of ContentMolecular Enzymology
TERTIARY LEVEL BIOLOGY
A series covering selected areas of biology at advanced
undergraduate level. While designed specifically for course
options at this level within Universities and
Polytechnics, the series will be of great value to
specialists and research workers in other fields who require
a knowledge of the essentials of a subject.
Titles in the series:
Experimentation in Biology Ridgman
Methods in Experimental Biology Ralph
Visceral Muscle Huddart and Hunt
Biological Membranes Harrison and Lunt
Comparative Immunobiology Manning and Turner
Water and Plants Meidner and Sheriff
Biology of Nematodes Croll and Matthews
An Introduction to Biological Rhythms Saunders
Biology of Ageing Lamb
Biology of Reproduction Hogarth
An Introduction to Marine Science Meadows and Campbell
Biology of Fresh Waters Maitland
An Introduction to Developmental Biology Ede
Physiology of Parasites Chappell
Neurosecretion Maddrell and Nordmann
Biology of Communication Lewis and Gower
Population Genetics Gale
Structure and Biochemistry of
Cell Organelles Reid
Developmental Microbiology Peberdy
Genetics of Microbes Bainbridge
Biological Functions of Carbohydrates Candy
Endocrinology Goldsworthy, Robinson
and Mordue
The Estuarine Ecosystem McLusky
Animal Osmoregulation Rankin and Davenport
TERTIARY LEVEL BIOLOGY
Molecular Enzymology
CHRISTOPHER w. WHARTON, B.Sc., Ph.D.
Lecturer in Biochemistry
University of Birmingham
and
ROBERT EISENTHAL, A.B., Ph.D.
Senior Lecturer in Biochemistry
Universityof Bath
Springer Science+
Business Media, LLC
Blackie & Son Limited
Bishopbriggs
Glasgow G64 2NZ
Furnival House
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© 1981 C. W. Wharton and R. Eisenthal
First published 1981
Softcover reprint of the hardcover 1s t edition 1981
AIIr ights reserved.
No part of this publication may be reproduced,
stored in a retrieval system, or transmitted,
in any form or by any means,
electronic, mechanical, recording or otherwise,
without prior permission of the Publishers
British Library Cataloguing in Publication Data
Wharton, Christopher W.
Molecular enzymology.- (Tertiary level biology)
1. Enzymes
1. Title II. Eisenthal, Robert. III. Series
574.19'25 QP601
ISBN 978-1-4615-8534-3 ISBN 978-1-4615-8532-9 (eBook)
DOI 10.1007/978-1-4615-8532-9
Filmset by Advanced Filmsetters (Glasgow) Ltd.
Preface
MOLECULAR ENZYMOLOGY, BECAUSE OF ITS CHEMICAL AND MATHEMATICAL
content, is often regarded as a formidable and forbidding topic by
undergraduates on a biology or biochemistry course, As a result of
teaching enzymology to undergraduates for a number of years, we
recognize the areas which appear to cause the most common difficulties in
conceptual understanding. We feel that a book treating those areas by
means of a logical approach carefully developed from basic principles fills
a gap in the multiplicity of enzymology texts currently available. In writing
this book we h;:lVe had in mind the needs of Honours Biochemistry
students, in particular those who may take a special interest in
enzymology. The text covers the main bulk of the material required in the
second and third years of such courses. In addition, those taking courses in
Biological Chemistry may well find the book to be of central interest.
The book begins with a description of the fundamentals of catalysis,
illustrating these with simple chemical reactions which may be supposed to
serve as models of catalytic processes. Protein structure is discussed in
terms of the fundamental forces which determine the shape and dynamic
behaviour of protein molecules. The approach emphasizes those features
thought to be most intimately involved in the catalytic function of enzyme
molecules, and is illustrated with specific examples.
Although a number of excellent texts dealing specifically with enzyme
kinetics have appeared over the past four years, the subject has not been
adequately treated in textbooks which purport also to cover the
mechanism of enzyme catalysis. We have therefore emphasized in our
chapters on enzyme kinetics the asp,ects required for a sound under
standing of enzymic catalysis. The fundamental kinetic equations which
describe both the transient and the steady state are fully derived, and
v
VI PREFACE
the equations are extended to the description of inhibition, pH
dependence, multisubstrate kinetics and cooperativity. Stress is laid upon
the practical aspects of methods of assay and analysis and interpretation of
kinetic data.
In discussing the classical mechanisms of reactions involving coenzymes,
the prime focus is upon the coenzyme chemistry but attention is paid to the
role of the enzyme molecule in the catalytic process. The penultimate
chapter treats in depth several enzymes whose mechanisms have been
extensively studied - here detailed consideration is given to the role played
by the amino-acid side-chain assemblies at the active centre. Our choice of
examples is governed by the need to expose the underlying principles in the
clearest possible fashion.
Recent advances in our understanding of the origin and evolution of
enzyme catalytic power are discussed. Model systems based on polymers
and macrocycles are described in an attempt to fuse the structural and
mechanistic aspects of enzymic catalysis. The bibliographic references
include both leading review articles and seminal papers from the primary
literature and should provide a comprehensive basis for further reading.
We are grateful to Keith Brocklehurst, Athel Cornish-Bowden, Mike
Danson, Roger Harrison, Bruno Orsi and Keith Tipton for their helpful
comments and to Pat Wharton for preparing many of the diagrams.
C.W.W.
R.E.
Contents
Chapter 1 THE NATURE OF CATALYSIS 1
Introduction-chemical reaction rates and their description
The relationship between free energy diagrams and rate constants
The principle of microscopic reversibility
Limiting factors in reaction rates
The nature of catalysis
The general nature of enzyme catalysis
Chapter 2 CHEMICAL CAT AL YSIS 14
Nucleophilic catalysis
General catalysis
Reactivity correlations in catalysis
The Hammond postulate
The Hammett equation
Curved free energy relationships
Application of structure~reactivity correlations in enzyme
chemistry
Kinetic ambiguity
Isotope effects
Approximation, internal rotation and strain
Rotamer distribution
Ground state strain
Some examples of simple compounds that demonstrate thp
approximation, internal rotation and strain effects
Conclusions
Chapter 3 PROTEIN STRUCTURE 44
Hydrogen bonds
Hydrophobic interactions
Electrostatic interactions
The structure of pancreatic ribonuclease (RNase)
The contribution of X-ray crystallography to the determination of
the structure of ribonuclease and other enzymes
The conformational dynamics of ribonuclease
Oligomeric proteins
vii
VIll CONTENTS
Chapter 4 SIMPLE ENZYME KINETICS 73
Michaelis-Menten equation
Maximum velocity and kca!
Michaelis constant
Units of enzyme activity
Presentation of kinetic data
The steady state
The enzyme-substrate complex
Reversible reactions
Equilibrium and the Haldane relationship
Substrate inhibition
Analysis of the progress curve
Summary
Chapter 5 COENZYMES 99
Adenosine triphosphate (ATP)
Nicotinamide adenine dinucleotide (NAD)
Flavin nucleotides (FAD)
Coenzyme A (CoA)
Thiamine pyrophosphate (TPP)
Pyridoxal phosphate
Chapter 6 EFFECTS OF INHIBITORS AND pH 120
Inhibitors-reversible and irreversible inhibitions, simple
reversible inhibition, competitive inhibition, uncompetitive
inhibition, mixed and non-competitive inhibition, other plotting
methods, competitive substrates, non-productive binding,
transition-state analogues, partial inhibition
Effects of pH --pH profiles, analysis of pH profiles, interpretation
of pK values from pH profiles, effect of substrate on pH profiles,
effects of buffers, ions and solvent, other effects
Chapter 7 COMPLEX KINETICS AND
COOPERAT IVITY 169
Complex kinetics-bisubstrate kinetics, random rapid equilibrium
mechanism, compulsory order mechanism, ping-pong (substituted
enzyme) mechanism, analysis of rate equations, significance of the
kinetic constants, plotting results, product inhibition, equilibrium
isotope exchange
Cooperativity-the Hill equation, the Adair equation, the Monod,
Wyman and Changeux (MWC) model, the Koshland, Nemethy
and Filmer (KNF) model, distinction between the MWC and
KNF models, heterotropic (allosteric) interactions, half-of-the
sites reactlVlty, other models of cooperativity -subunit
depolymerization, kinetic models of cooperativity, the Rabin
mechanism, the mnemonical mechanism, other situations in which
apparent cooperativity may be observed, molecular mechanisms
of cooperativity
CONTENTS IX
Chapter 8 THE ANALYSIS OF ENZYME 204
MECHANISMS BY MEANS OF FAST
REACTION TECHNIQUES
Analysis of the simple mechanism
Flow techniques of following fast reactions
Burst kinetics
Rapid scanning spectrophotometry
Perturbation methods
Cryogenic techniques
Chapter 9 ENZYME MECHANISMS 220
The serine proteases-the chymotrypsin-catalysed hydrolysis of p
nitrophenyl acetate, active site directed inhibition, active site
titration, the catalysis of the hydrolysis of specific substrates,
nucleophile partitioning, the pH-dependence of a-chymotrypsin
catalysed hydrolyses, transition state analogues, X-ray
crystallographic studies, the chemical mechanism, proton
inventories, the origin of the catalytic rate enhancement
The thiol proteases-inhibition and titration of the thiol proteases,
the specificity of papain, the pH-dependence of papain-catalysed
hydrolyses, the X-ray structure of papain, the reactive protonic
form of the enzyme, the catalytic mechanism of papain
Ribonuclease (RNase)-the pH-dependence of RNase catalysis,
inhibition of RNase
Glyceraldehyde 3-phosphate dehydrogenase
Lactate and alcohol dehydrogenases
Triose phosphate isomerase
Glucokinase
The origin and evolution of enzyme catalytic power
Synthetic models of enzymic catalysis
Chapter 10 PRACTICAL ENZYME KINETICS 275
Graphical display of kinetic data
Calculation of Km and V
Systematic deviation from Michaelis-Menten kinetics
Range and spacing of So
Importance of initial rates
Why does the rate decrease?
Estimation of initial rates
Techniques of assay-direct assays, coupled assays, spectrophoto
metry, fluorimetry, pH-potentiometry, polarography and the
oxygen electrode, radiochemical methods
Derivation of rate equations
BIBLIOGRAPHY 310
INDEX 319
