Fundamentals of Enzyme Kinetics

Principal Symbols Used in this Book1 Basic Principles of Chemical Kinetics 1.1 Order of a Reaction 1.2 Determination of the Order of a Reaction 1.3 Dimensions of Rate Constants 1.4 Reversible Reactions 1.5 Determination of First-Order Rate Constants 1.6 Influence of Temperature on Rate Constants 1.7 Transition-State Theory Problems2 Introduction to Enzyme Kinetics 2.1 Early Studies: The Idea of an Enzyme-Substrate Complex 2.2 Michaelis-Menten Equation 2.3 Steady-State Treatment 2.4 Validity of the Steady-State Assumption 2.5 Graphical Representation of the Michaelis-Menten Equation 2.6 Reversible Michaelis-Menten Mechanism 2.7 Product Inhibition 2.8 Integrated Michaelis-Menten Equation Problems3 Practical Considerations 3.1 Purification of Enzymes 3.2 Enzyme Assays 3.3 Coupled Assays 3.4 Protein Determination 3.5 Presentation of Results of a Purification 3.6 Detecting Enzyme Inactivation 3.7 Experimental Design: Choice of Substrate Concentrations 3.8 Choice of Ph, Temperature and Other Conditions 3.9 Use of Replicate Observations 3.10 Treatment of Ionic Equilibria Problems4 How to Derive Steady-State Rate Equations 4.1 King-Altman Method 4.2 Modifications to the King-Altman Method 4.3 Cha's Method for Reactions Containing Steps at Equilibrium 4.4 Analyzing Mechanisms by Inspection Problems5 Inhibitors and Activators 5.1 Reversible and Irreversible Inhibitors 5.2 Competitive Inhibition 5.3 Mixed Inhibition 5.4 Uncompetitive Inhibition 5.5 Plotting Inhibition Results 5.6 Inhibition by a Competing Substrate 5.7 Activation 5.8 Hyperbolic Inhibition and Activation 5.9 Design of Inhibition Experiments 5.10 Non-Productive Binding 5.11 Substrate Inhibition 5.12 Chemical Modification as a Means of Identifying Essential Groups Problems6 Two-Substrate Reactions 6.1 Introduction 6.2 Types of Mechanism 6.3 Rate Equations 6.4 Initial-Velocity Measurements in the Absence of Products 6.5 Substrate Inhibition 6.6 Product Inhibition 6.7 Design of Experiments 6.8 Isotope Exchange 6.9 Reactions with Three or More Substrates Problems7 Effects of pH and Temperature on Enzymes 7.1 pH and Enzyme Kinetics 7.2 Acid-Base Properties of Proteins 7.3 Ionization of a Dibasic Acid 7.4 Effect of pH on Enzyme Kinetic Constants 7.5 Ionization of the Substrate 7.6 More Complex pH Effects 7.7 Temperature Dependence of Enzyme-Catalyzed Reactions Problems8 Control of Enzyme Activity 8.1 Necessity for Metabolic Control 8.2 Binding of Oxygen to Hemoglobin 8.3 Hill Equation 8.4 Adair Equation 8.5 Definition of Co-operativity 8.6 Induced Fit 8.7 Symmetry Model of Monod, Wyman and Changeux 8.8 Sequential Model of Koshland, Nꮥthy and Filmer 8.9 Other Models for Co-operativity at Equilibrium 8.10 Kinetic Models of Co-operativity Problems9 Fast Reactions 9.1 Limitations of Steady-State Measurements 9.2 Active-Site Titration: 'Burst' Kinetics 9.3 Flow Methods 9.4 Relaxation Methods 9.5 Transient-State Kinetics of Systems Far from Equilibrium 9.6 Simplification of Complex Mechanisms 9.7 Kinetics of Systems Close to Equilibrium Problems10 Estimation of Kinetic Constants 10.1 Cautionary Note 10.2 Least-Squares Fit to the Michaelis-Menten Equation 10.3 Statistical Aspects of the Direct Linear Plot 10.4 Precision of Km and V Estimates 10.5 Examination of Residuals ProblemsReferencesSolutions to ProblemsIndex