Copeland R.A. Enzymes. A Practical Introduction to Structure, Mechanism, and Data Analysis

2nd edition. Wiley-VCH, Inc. 2000. - 390 p.
This book is intended to serve as a mechanism for those new to the field of enzymology to develop a reasonable understanding of the science and experimental methods, allowing them to competently begin laboratory studies with enzymes.

Contents

Preface

Acknowledgments

Preface to the First Edition

A Brief History of Enzymology

Enzymes in Antiquity
Early Enzymology
The Development of Mechanistic Enzymology
Studies of Enzyme Structure
Enzymology Today
Summary
References and Further Reading

Chemical Bonds and Reactions in Biochemistry

Atomic and Molecular Orbitals
Thermodynamics of Chemical Reactions
Acid—Base Chemistry
Noncovalent Interactions in Reversible Binding
Rates of Chemical Reactions
Summary
References and Further Reading

Structural Components of Enzymes

The Amino Acids
The Peptide Bond
Amino Acid Sequence or Primary Structure
Secondary Structure
Tertiary Structure
Subunits and Quateary Structure
Cofactors in Enzymes
Summary
References and Further Reading

Protein?Ligand Binding Equilibria

The Equilibrium Dissociation Constant, K_d
The Kinetic Approach to Equilibrium
Binding Measurements at Equilibrium
Graphic Analysis of Equilibrium Ligand Binding Data
Equilibrium Binding with Ligand Depletion (Tight Binding Interactions)
Competition Among Ligands for a Common Binding Site
Experimental Methods for Measuring Ligand Binding
Summary
References and Further Reading

Kinetics of Single-Substrate Enzyme Reactions

The Time Course of Enzymatic Reactions
Effects of Substrate Concentration on Velocity
The Rapid Equilibrium Model of Enzyme Kinetics
The Steady State Model of Enzyme Kinetics
The Significance of k_cat and K_m
Experimental Measurement of k_cat and K_m
Other Linear Transformations of Enzyme Kinetic Data
Measurements at Low Substrate Concentrations
Deviations from Hyperbolic Kinetics
Transient State Kinetic Measurements
Summary
References and Further Reading

Chemical Mechanisms in Enzyme Catalysis

Substrate—Active Site Complementarity
Rate Enhancement Through Transition State Stabilization
Chemical Mechanisms for Transition State Stabilization
The Serine Proteases: An Illustrative Example
Enzymatic Reaction Nomenclature
Summary
References and Further Reading

Experimental Measures of Enzyme Activity

Initial Velocity Measurements
Detection Methods
Separation Methods in Enzyme Assays
Factors Affecting the Velocity of Enzymatic Reactions
Reporting Enzyme Activity Data
Enzyme Stability
Summary
References and Further Reading

Reversible Inhibitors

Equilibrium Treatment of Reversible Inhibition
Modes of Reversible Inhibition
Graphic Determination of Inhibitor Type
Dose—Response Curves of Enzyme Inhibition
Mutually Exclusive Binding of Two Inhibitors
Structure—Activity Relationships and Inhibitor Design
Summary
References and Further Reading

Tight Binding Inhibitors

Identifying Tight Binding Inhibition
Distinguishing Inhibitor Type for Tight Binding Inhibitors
Determining K_i for Tight Binding Inhibitors
Use of Tight Binding Inhibitors to Determine Active Enzyme Concentration
Summary
References and Further Reading

Time-Dependent Inhibition

Progress Curves for Slow Binding Inhibitors
Distinguishing Between Slow Binding Schemes
Distinguishing Between Modes of Inhibitor Interaction with Enzyme
Determining Reversibility
Examples of Slow Binding Enzyme Inhibitors
Summary
References and Further Reading

Enzyme Reactions with Multiple Substrates

Reaction Nomenclature
Bi Bi Reaction Mechanisms
Distinguishing Between Random and Compulsory Ordered Mechanisms by Inhibition Patte /
Isotope Exchange Studies for Distinguishing Reaction Mechanisms
Using the King—Altman Method to Determine Velocity Equations
Summary
References and Further Reading

Cooperativity in Enzyme Catalysis

Historic Examples of Cooperativity and Allostery in Proteins
Models of Allosteric Behavior
Effects of Cooperativity on Velocity Curves
Sigmoidal Kinetics for Nonallosteric Enzymes
Summary
References and Further Reading

Appendix I Suppliers of Reagents and Equipment for Enzyme Studies

Appendix II Useful Computer Software and Web Sites for Enzyme Studies

Index