Comprehensive Enzyme Kinetics

Transcription

1 Comprehensive Enzyme Kinetics

2 Comprehensive Enzyme Kinetics Vladimir Leskovac University of Novi Sad KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

3 ebook ISBN: Print ISBN: Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow Print 2003 Kluwer Academic/Plenum Publishers New York All rights reserved No part of this ebook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: and Kluwer's ebookstore at:

4 Preface Welcome to your study of enzyme kinetics, the subject that underlies all enzymology, which in turn underlies all aspects of biochemistry. This text will give you an introduction to a wide range of topics that constitute the modern enzyme kinetics. This textbook is directed at graduate students in biochemistry, chemistry, and life sciences, for advanced courses in enzyme kinetics, enzymology, and enzyme chemistry. For this reason, the whole book is organized in a systematic and scholarly fashion. It is unlikely that the student will be expected to cover everything in the text, but in a later career she or he may find it an invaluable reference for topics that are needed in practice. The concepts, definitions and detailed algebra of enzyme kinetics are laid out in accurate detail. For that reason, this textbook can also serve as a handbook for enzyme kinetics for research workers in the field. The research worker will find it a useful source, which can be used for solving the daily experimental problems in the laboratory. The preparation of the manuscript for this book was under the constant surveillance of W. Wallace Cleland, Professor of Chemical Science at the University of Wisconsin in Madison, and one of the founders of modern enzyme kinetics. Without his help and advice, this book would not be possible. Several versions of the manuscript were constantly corrected and improved by Svetlana Professor of Biochemistry at the University of Novi Sad. The final version of the manuscript was corrected by Dexter B. Northrop, Professor of Biochemistry at the University of Wisconsin in Madison, by Richard L. Schowen, Professor of Chemistry and Biochemistry at the University of Kansas in Lawrence, and by Bryce V. Plapp, Professor of Biochemistry at the University of Iowa in Iowa City. The writing and the technical preparation of this book was supported by able computer engineers in Novi Sad, who provided the software, the hardware, and the maintenance of our computer facilities. The Faculty of Technology at the University of Novi Sad provided the logistics and the financial support for the preparation of the manuscript. Also, I owe my thanks to the staff of the Kluwer Academic/Plenum Publishers, for their constant help and patience during the preparation of this book. Alden Bookset was responsible for an excellent typesetting of this book. Finally, as the former disciple of Professor Gerhard Pfleiderer, I dedicate this book to him, in recognition of his germinal and indelible contributions to the field of enzyme structure and function. Vladimir Leskovac v

5 Contents 1. Introduction Enzyme Structure The Active Site Chemical Kinetics Molecularity and Order of Reaction Determination of the Order of Reaction Determination of Rate Constants Reversible Reactions Consecutive Reactions Influence of Temperature on Rate Constants Properties of Water Ionization of Acids Dimensions and Dimensional Analysis Kinetics of Monosubstrate Reactions 3.1 Work of Micnaelis and Menten 3.2 Steady-State Approximation 3.3 Reversible Mechanism with One Central Complex 3.4 Reversible Mechanism with Two Central Complexes 3.5 Equilibrium Constant and the Haldane Relationship 3.6 Rate Equations in Coefficient Form 3.7 Enzyme Distribution Equations 3.8 Hyperbolic Nature of the Michaelis Menten Equation 3.9 Significance of Kinetic Parameters 3.10 Graphical Presentation of Data 3.11 Dimensional Analysis Derivation of Rate Equations 4.1 Velocity Equations for Rapid Equilibrium Systems 4.2 Net Rate Constant Method vii

6 viii CONTENTS Method of King and Altman The Method of Cha The Systematic Approach Comparison of Different Methods Linear Inhibition Classification of Inhibitors Competitive Inhibition Noncompetitive Inhibition Uncompetitive Inhibition Characteristics of Linear Inhibition Dead-End Inhibition in Steady-State Bisubstrate Systems Inhibition bv a Mixture of Two Inhibitors Hyperbolic and Parabolic Inhibition Hyperbolic Inhibition in Monosubstrate Reactions Determination of Kinetic Constants in Hyperbolic Inhibition Variations of Hyperbolic Inhibition in Monosubstrate Reactions Hyperbolic Inhibition in Bisubstrate Reactions Parabolic Inhibition in Monosubstrate Reactions Nomenclature of Double Reciprocal Plots in the Presence of Inhibitors Enzyme Activation Nonessential Activation Essential Activation Kinetics of Rapid Equilibrium Bisubstrate Reactions Nomenclature Rapid Equilibrium Ordered System Rapid Equilibrium Random System Product Inhibition in a Rapid Equilibrium Ordered Bi Bi System Product Inhibition in a Rapid Equilibrium Random Bi Bi System Rapid Equilibrium RandomBiBiSystem with a Dead-End EBQ Complex A Random Model with Two Dead-End Complexes Diagnostics of Rapid Equilibrium Systems

7 CONTENTS ix 9. Steady-State Kinetics of Bisubstrate Reactions Nomenclature Ordered Bi Bi System Theorell Chance Mechanism Ordered Uni Bi and Ordered Bi Uni Systems Ping Pong Bi Bi Mechanism Steady-State Random Bi Uni Mechanism Isomerization of Transitory Complexes and Stable Enzyme Forms Kinetic Analysis of Bisubstrate Mechanisms Prediction of Initial Velocity Patterns Product Inhibition Analysis of Intersection Points Special Relationships between the Constants Complex Bisubstrate Mechanisms with a Central Ternary Complex Complex Ping Pong Mechanisms Examples of Enzyme Bisubstrate Mechanisms Substrate Inhibition and Mixed Dead-End and Product Inhibition Rational Polynomials Substrate Inhibition Non-Productive Binding Mixed Dead-End and Product Inhibition Summary of Inhibition Types Trisubstrate Mechanisms Prediction of Reaction Sequences in Trisubstrate Mechanisms Rate Equations in the Absence of Products Full Rate Equations for Ter Bi Systems Full Rate Equations for Ter Ter Systems Product Inhibition in Trisubstrate Mechanisms Examples of Enzyme Trisubstrate Mechanisms Cooperative and Allosteric Effects 13.1 Cooperative Versus Allosteric Effects 13.2 Noncooperative Binding 13.3 Positive Cooperativity

8 x CONTENTS Negative Cooperativity Monod, Wyman. and Changeux Model (MWC Model) Application of the MWC Model to Enzymes Koshland, Nemethy, and Filmer Models (KNF Models) Comparison of Various Models The ph Dependence of Enzyme Catalysis Introduction Dissociation of Dibasic Acids Effects of ph on Kinetics of Monosubstrate Reactions Effects of ph on Kinetics of Bisubstrate Reactions ph Profiles Interpretation of ph Profiles Summary of Procedures to Follow the ph Studies Effects of Temperature on Enzyme Reactions Free Energy Profiles Separation and Estimation of Rate Constants Heat of lonization of Amino Acid Side Chains in Enzymes Solvent Perturbation Isotope Exchange Principles of Isotope Exchange Isotopic Exchange at Chemical Equilibrium in Sequential Mechanisms Specific Examples of Sequential Mechanisms Sequential Mechanisms away from Equilibrium Isotopic Exchange in Ping Pong Mechanisms Abortive Complex Formation Kinetic Isotope Effects Origin of Isotope Effects Tritium versus Deuterium Kinetic Isotope Effects Kinetic Isotope Effects on and in Enzymatic Reactions Determination of Kinetic Isotone Effects Kinetic Mechanism from the Variation of Substrates and Products Kinetic Mechanism from the Variation of Kinetic Isotope Effects with ph

9 CONTENTS xi Isotope Trapping Heavy-Atom Kinetic Isotope Effects Secondary Isotope Effects Solvent Isotope Effects Statistical Analysis of Initial Rate and Binding Data Statistics Analysis of Initial Rate Data Model Discrimination Integrated Michaelis Menten Equation Subject Index 421