Fundamentals of Enzyme Kinetics E-Book

Athel Cornish-Bowden

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 1141

Veröffentlichungsjahr: 2013

Beschreibung

Now in its fourth edition, this textbook is one of the few titles worldwide to cover enzyme kinetics in its entire scope and the only one to include its implications for bioinformatics and systems biology. Multi-enzyme complexes and cooperativity are therefore treated in more detail than in any other textbook on the market.
The respected and well known author is one of the most experienced researchers into the topic and writes with outstanding style and didactic clarity. As with the previous editions, he presents here steady-state kinetics and fast reactions, supplementing each chapter with problems and solutions. For the first time, this edition features a companion website providing all figures in colour
www.wiley-vch.de/home/fundenzykinet

Leseprobe

Contents

Preface

Chapter 1 Basic Principles of Chemical Kinetics

1.1 Symbols, terminology and abbreviations

1.2 Order of a reaction

1.3 Dimensions of rate constants

1.4 Reversible reactions

1.5 Determination of first-order rate constants

1.6 The steady state

1.7 Catalysis

1.8 The influence of temperature and pressure on rate constants

Chapter 2 Introduction to Enzyme Kinetics

2.1 The idea of an enzyme–substrate complex

2.2 The Michaelis–Menten equation

2.3 The steady state of an enzyme-catalyzed reaction

2.4 Specificity

2.5 Validity of the steady-state assumption

2.6 Graphs of the Michaelis–Menten equation

2.7 The reversible Michaelis–Menten mechanism

2.8 Product inhibition

2.9 Integration of enzyme rate equations

Chapter 3 “Alternative” enzymes

3.1 Introduction

3.2 Artificial enzymes

3.3 Site-directed mutagenesis

3.4 Chemical mimics of enzyme catalysis

3.5 Catalytic RNA

3.6 Catalytic antibodies

Chapter 4 Practical Aspects of Kinetics

4.1 Enzyme assays

4.2 Detecting enzyme inactivation

4.3 Experimental design

4.4 Treatment of ionic equilibria

Chapter 5 Deriving Steady-state Rate Equations

5.1 Introduction

5.2 The principle of the King–Altman method

5.3 The method of King and Altman

5.4 The method of Wong and Hanes

5.5 Modifications to the King–Altman method

5.6 Reactions containing steps at equilibrium

5.7 Analyzing mechanisms by inspection

5.8 A simpler method for irreversible reactions

5.9 Derivation of rate equations by computer

Chapter 6 Reversible Inhibition and Activation

6.1 Introduction

6.2 Linear inhibition

6.3 Plotting inhibition results

6.4 Multiple inhibitors

6.5 Relationship between inhibition constants and the concentration for 50% inhibition

6.6 Inhibition by a competing substrate

6.7 Enzyme activation

6.8 Design of inhibition experiments

6.9 Inhibitory effects of substrates

Chapter 7 Tight-binding and Irreversible Inhibitors

7.1 Tight-binding inhibitors

7.2 Irreversible inhibitors

7.3 Substrate protection experiments

7.4 Mechanism-based inactivation

7.5 Chemical modification as a means of identifying essential groups

7.6 Inhibition as the basis of drug design

7.7 Delivering a drug to its target

Chapter 8 Reactions of More than One Substrate

8.1 Introduction

8.2 Classification of mechanisms

8.3 Rate equations

8.4 Initial-rate measurements in the absence of products

8.5 Substrate inhibition

8.6 Product inhibition

8.7 Design of experiments

8.8 Reactions with three or more substrates

Chapter 9 Use of Isotopes for Studying Enzyme Mechanisms

9.1 Isotope exchange and isotope effects

9.2 Principles of isotope exchange

9.3 Isotope exchange at equilibrium

9.4 Isotope exchange in substituted-enzyme mechanisms

9.5 Nonequilibrium isotope exchange

9.6 Theory of kinetic isotope effects

9.7 Primary isotope effects in enzyme kinetics

9.8 Solvent isotope effects

Chapter 10 Effect of pH on Enzyme Activity

10.2 Acid–base properties of proteins

10.3 Ionization of a dibasic acid

10.4 Effect of pH on enzyme kinetic constants

10.5 Ionization of the substrate

10.6 “Crossed-over” ionization

10.7 More complicated pH effects

Chapter 11 Temperature Effects on Enzyme Activity

11.1 Temperature denaturation

11.2 Irreversible denaturation

11.3 Temperature optimum

11.4 Application of the Arrhenius equation to enzymes

11.5 Entropy–enthalpy compensation

Chapter 12 Regulation of Enzyme Activity

12.1 Function of cooperative and allosteric interactions

12.2 The development of models for cooperativity

12.3 Analysis of binding experiments

12.4 Induced fit

12.5 The symmetry model of Monod, Wyman and Changeux

12.6 Comparison between the principal models of cooperativity

12.7 The sequential model of Koshland, Némethy and Filmer

12.8 Association-dissociation models of cooperativity

12.9 Kinetic cooperativity

Chapter 13 Multienzyme Systems

13.1 Enzymes in their physiological context

13.2 Metabolic control analysis

13.3 Elasticities

13.4 Control coefficients

13.5 Properties of control coefficients

13.6 Relationships between elasticities and control coefficients

13.7 Response coefficients: the partitioned response

13.8 Control and regulation

13.9 Mechanisms of regulation

13.10 Computer modeling of metabolic systems

13.11 Biotechnology and drug discovery

Chapter 14 Fast Reactions

14.1 Limitations of steady-state measurements

14.2 Product release before completion of the catalytic cycle

14.3 Experimental techniques

14.4 Transient-state kinetics

Chapter 15 Estimation of Kinetic Constants

15.1 Data analysis in an age of kits

15.2 The effect of experimental error on kinetic analysis

15.3 Least-squares fit to the Michaelis–Menten equation

15.4 Statistical aspects of the direct linear plot

15.5 Precision of estimated kinetic parameters

APPENDIX: Standards for Reporting Enzymology Data

A1 Introduction

A2 General information

A3 Kinetic information

A4 Organism-related information

A5 What you can do

Solutions and Notes to Problems

Index

Related Titles

Alberty, R. A.

Enzyme Kinetics

Rapid-Equilibrium Applications of Mathematica. includes CD-ROM, Volume 53

2011

ISBN: 978-0-470-63932-0

Bisswanger, H.

Enzyme Kinetics

Principles and Methods

2008

ISBN: 978-3-527-31957-2

Bisswanger, H.

Practical Enzymology

2011

ISBN: 978-3-527-32076-9

Voet, D. J., Voet, J. G.

Biochemistry

2011

ISBN: 978-0-470-57095-1

Pratt, C. W., Cornely, K.

Essential Biochemistry

2010

ISBN: 978-0-470-50477-2

The Author

Dr. Athel Cornish-Bowden

Bioénergétique et Ingénierie des Protéines, CNRS

31, Chemin Joseph Aiguier

13402 Marseille Cedex 20

France

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty can be created or extended by sales representatives or written sales materials. The Advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

Library of Congress Card No.:

applied for

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.

Bibliographic information published by the Deutsche Nationalbibliothek

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d-nb.de>.

© 2012 Wiley-VCH Verlag & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany

Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical, and Medical business with Blackwell Publishing.

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Composition Uwe Krieg, Berlin

Cover Design Adam Design, Weinheim

Print ISBN: 978-3-527-33074-4

Preface to the Fourth Edition

It was said of the great statistician R. A. Fisher that whenever he introduced a result with the words “it can easily be shown that…” one could be sure that two or three hours of hard work would be in store for anyone wishing to verify it. As a student I thought that many authors used this formula as a way to avoid explaining things that they could not explain. I hasten to add that in Fisher’s case I am sure there was no lack of ability, though there may have been a lack of appreciation of the difficulties that his readers had. When I was writing the earliest version of this book, therefore, I resolved never to claim that anything was easy unless I was quite sure that it was. In the 35 years that have passed since then I believe I have kept this resolution, though I have often had to revise my views about what was simple enough to be left unexplained. Above all I have striven for clarity, being guided by a slogan from Keith Laidler: “Correctness, cogency, clarity: these three, but the greatest of these is clarity”. Errors can be corrected, weak arguments can be strengthened, but lack of clarity leaves a fog that may take years to dispel.

The emphasis throughout is on understanding enzyme kinetics, not on covering every aspect of the subject in an encyclopedic style. So I have preferred to describe the principles that will allow readers to proceed as far as they want in any direction. In the words of Kuan-tzu (as quoted by Parzen): “If you give a man a fish, he will have a single meal; if you teach him how to fish, he will eat all his life”.

I make no apology for continuing to illustrate concepts with abundant graphs, including the straight-line graphs that biochemists have used for three-quarters of a century, although it is sometimes argued that the appearance of computers on every desk has made graphical methods obsolete. Professional statisticians who really know and understand data analysis think differently; for example, Chambers and co-workers wrote

There is no statistical tool that is as powerful as a well-chosen graph. Our eye–brain system is the most sophisticated information processor ever developed, and through graphical displays we can put this system to good use to obtain deep insight into the structure of data.

There is little to “see” in a biochemical experiment and almost all our information comes at second hand from instruments, so it is essential to convert it into something visible. At the same time judicious use of the computer is equally necessary—not just graphs, not just computation, but both, in partnership—and in this spirit I have not only retained but have expanded the final chapter of the book, which has been a well received feature of the earlier editions.

Enzyme kinetics is not a topic that changes greatly from year to year, so why is a new edition needed? The text has of course been updated, with greater recognition of the importance of enzyme kinetics for biotechnology and drug development, and many recent literature references have been added. The major and most obvious change, however, is in the manner of presenting the information. There are more than three times as many figures as there were in the third edition, and the need for page-flipping has been virtually eliminated: not only figures and tables, but also references and notes, all appear as close as possible to the context in which they are mentioned; any that cannot appear on the same page opening where they are mentioned are never more than a page away. Here it is a pleasure to acknowledge the willingness of Wiley–VCH to allow the book to be laid out exactly as I wanted.

The original ancestor of this book was called Principles of Enzyme Kinetics, and appeared in 1976. Later I decided that the treatment needed to be made more elementary, and in 1979 the first edition of Fundamentals of Enzyme Kinetics had a new title to reflect the different emphasis. Over the years, however, much of the text that was dropped in 1979 has been put back, together with a significant amount of new material that is not particularly elementary. A case could be made, however, for reinstating the original title, or just calling it Enzyme Kinetics, but I have discarded this course in order not to give the impression that it is more different than it is from the third edition.

In this edition I have added numerous brief biographies of some of the scientists who created enzymology. Why? Will it help students to be better biochemists if they know that Maud Menten was a woman, that James Sumner was left-handed but had lost the use of his left hand in a childhood accident, or that Emil Fischer’s father considered him too stupid to be a businessman? Obviously not, but it will help them to understand that enzymology did not spring from nowhere, but was developed by real people with the same difficulties and hardships that people face today.

Acknowledgments

This edition has benefited greatly from the comments of many people who have read it all or in part: Dan Beard, Keith Brocklehurst, Marilú Cárdenas, Gilles Curien, Roy Daniel, Michael Danson, David Fell, Herbert Friedmann, Bob Goldberg, Brigitte Gontero, Jannie Hofmeyr, Peter Hughes, Marc Jamin, Carsten Kettner, Ana Ponces, Valdur Saks, Marius Schmidt, Keith Tipton, Chris Wharton. I have not followed all of their suggestions, and they are anyway not responsible for any faults that remain, but I have followed most of them, and I am extremely grateful for all of their comments.

I acknowledge with gratitude the Centre National de la Recherche Scientifique for giving me the possibility of continuing working as Directeur de RechercheÉmérite. It is likewise a pleasure also to acknowledge Dr. Bruno Guigliarelli, Director of the Laboratory of Ingénierie et Bioénergétique des Protéines of the Centre National de la Recherche Scientifique, and Dr. Marie-Thérèse Giudici-Orticoni, head of the group that I have joined since becoming Emeritus, both of them for their general support and for their success in creating a congenial working environment.

A different sort of acknowledgment is needed for Bob Alberty, still active at the age of 90 in the subject that he helped revolutionize in the 1950s. He first wrote to me in 1977, and I was delighted that my first effort to write a book about enzyme kinetics had found favor with a giant in the subject; subsequently he has given me much encouragement.

As mentioned already, the publishers were very cooperative in allowing a layout that would achieve my aim of making it as easy as possible to find insertions referred to in the text.

Marilú Cárdenas read all of the book in proof with me, and allowed numerous errors to be corrected. However, I owe her far more than that: as my wife as well as my collaborator (and originally my competitor in the field of hexokinase research), she has contributed in innumerable ways to my life during the past 30 years.

Corrections

It would be nice to think that there were no typographical or other errors in this book. Nice, yes, but if past experience is any guide, not very realistic, so a list of corrections will be maintained at http://bip.cnrs-mrs.fr/bip10/fek.htm.

Athel Cornish-BowdenMarseilles, July 2011

K. J. Laidler (1998) To Light such a Candle, Oxford University Press, Oxford

E. Parzen (1980) “Comment” American Statistician 34, 78–79

J. M. Chambers, W. S. Cleveland, B. Klein and P. A. Tukey (1983) Graphical Methods for Data Analysis, Wadsworth, Belmont, California

Chapter 1

Basic Principles of Chemical Kinetics

1.1 Symbols, terminology and abbreviations

This book follows as far as possible the recommendations of the International Union of Biochemistry and Molecular Biology. However, as these allow some latitude and in any case do not cover all of the cases that we shall need, it is useful to begin by noting some points that apply generally in the book. First of all, it is important to recognize that a chemical substance and its concentration are two different entities and need to be represented by different symbols. The recommendations allow square brackets around the chemical name to be used without definition for its concentration, so [glucose] is the concentration of glucose, [A] is the concentration of a substance A, and so on. In this book I shall use this convention for names that consist of more than a single letter, but it has the disadvantage that the profusion of square brackets can lead to forbiddingly complicated equations in enzyme kinetics (see some of the equations in Chapter 8, for example, and imagine how they would look with square brackets). Two simple alternatives are possible: one is just to put the name in italics, so the concentration of A is A, for example, and this accords well with the standard convention that chemical names are written in roman (upright) type and algebraic symbols are written in italics. However, experience shows that many readers barely notice whether a particular symbol is roman or italic, and so it discriminates less well than one would hope between the two kinds of entity. For this reason I shall use the lower-case italic letter that corresponds to the symbol for the chemical entity, so a is the concentration of A, for example. If the chemical symbol has any subscripts, these apply unchanged to the concentration symbol, so a0 is the concentration of A0, for example. Both of these systems (and others) are permitted by the recommendations as long as each symbol is defined when first used. This provision is satisfied in this book, and it is good to follow it in general, because almost nothing that authors consider obvious is perceived as obvious by all their readers. In the problems at the ends of the chapters, incidentally, the symbols may not be the same as those used in the corresponding chapters: this is intentional, because in the real world one cannot always expect the questions that one has to answer to be presented in familiar terms.

Chapter 8, pages 189–226

The policy regarding the use of abbreviations in this book can be stated very simply: there are no abbreviations in this book (other than in verbatim quotations and the index, which needs to include the entries readers expect to find). Much of the modern literature is rendered virtually unintelligible to nonspecialist readers by a profusion of unnecessary abbreviations. They save little space, and little work (because with modern word-processing equipment it takes no more than a few seconds to expand all of the abbreviations that one may have found it convenient to use during preparation), but the barrier to comprehension that they represent is formidable. A few apparent exceptions (like “ATP”) are better regarded as standardized symbols than as abbreviations, especially because they are more easily understood by most biochemists than the words they stand for.

1.2 Order of a reaction

1.2.1 Order and molecularity

Chemical kinetics as a science began in the middle of the 19th century, when Wilhelmy was apparently the first to recognize that the rate at which a chemical reaction proceeds follows definite laws, but although his work paved the way for the law of mass action of Waage and Guldberg, it attracted little attention until it was taken up by Ostwald towards the end of the century, as discussed by Laidler. Wilhelmy realized that chemical rates depended on the concentrations of the reactants, but before considering some examples we need to examine how chemical reactions can be classified.

One way is according to the molecularity, which defines the number of molecules that are altered in a reaction: a reaction A → P is unimolecular (sometimes called monomolecular), and a reaction A + B → P is bimolecular. One-step reactions of higher molecularity are extremely rare, if they occur at all, but a reaction A + B + C → P would be trimolecular (or termolecular). Alternatively one can classify a reaction according to its order, a description of its kinetics that defines how many concentration terms must be multiplied together to get an expression for the rate of reaction. Hence, in a the rate is proportional to one concentration; in a it is proportional to the product of two concentrations or to the square of one concentration; and so on.