Enzyme Kinetics

Hans Bisswanger was Professor at the Interfaculty Institute of Biochemistry at the University of Tübingen (Germany), where he has developed and taught for many years an intensive course on enzyme kinetics, enzyme technology and ligand binding. His scientific interest lies with structural and regulatory mechanisms of multi-enzyme complexes, thermophilic enzymes and the technical application of immobilized enzymes. He is the author of two well-known books on enzymology that have appeared in different languages and editions.

• Preface xiSymbols and Abbreviations xiiiIntroduction and Definitions xv1 Multiple Equilibria, Principles, and Derivations 11.1 General Considerations 11.2 Diffusion 21.3 Modes of Ligand Binding 41.4 Interaction between Macromolecules and Ligands 61.4.1 Binding Constants 61.4.2 Binding to a Single Site 71.5 Binding to Identical Independent Sites 71.5.1 General Binding Equation 71.5.2 Graphic Representations of the Binding Equation 131.5.2.1 Direct and Linear Diagrams 131.5.2.2 Analysis of Binding Data from Spectroscopic Titrations 151.5.3 Binding of Different Ligands, Competition 181.5.4 Noncompetitive Binding 211.6 Binding to Nonidentical, Independent Sites 23References 252 Cooperativity and Allosteric Enzymes 272.1 Binding to Interacting Sites 272.1.1 The Hill Equation 272.1.2 The Adair Equation 292.1.3 The Pauling Model 322.2 Allosteric Enzymes 322.2.1 The Symmetry or Concerted Model 332.2.2 The SequentialModel and Negative Cooperativity 382.2.3 Analysis of Cooperativity 422.2.4 Physiological Aspects of Cooperativity 442.2.5 Examples of Allosteric Enzymes 462.2.5.1 Hemoglobin 462.2.5.2 Aspartate Transcarbamoylase 482.2.5.3 Aspartokinase 492.2.5.4 Phosphofructokinase 502.2.5.5 Allosteric Regulation of the Glycogen Metabolism 502.2.5.6 Membrane-Bound Enzymes and Receptors 502.3 Binding to Nonidentical, Interacting Sites 51References 523 FromReaction Order to the Michaelis–Menten Law: Fundamental Relationships of Enzyme Kinetics 553.1 Reaction Order 553.1.1 First-Order Reactions 563.1.2 Second-Order Reactions 573.1.3 Zero-Order Reactions 583.2 Steady-State Kinetics and the Michaelis–Menten Equation 583.2.1 Derivation of the Michaelis–Menten Equation 583.3 Analysis of Enzyme Kinetic Data 623.3.1 Graphic Representations of the Michaelis–Menten Equation 623.3.1.1 Direct and Semilogarithmic Representations 623.3.1.2 Direct Linear Plots 683.3.1.3 LinearizationMethods 703.3.2 Analysis of Progress Curves 723.3.2.1 Integrated Michaelis–Menten Equation 733.3.2.2 Determination of Reaction Rates 753.3.2.3 Graphic Methods for Rate Determination 773.3.2.4 Graphic Determination of True Initial Rates 793.4 Reversible Enzyme Reactions 803.4.1 Rate Equation for Reversible Enzyme Reactions 803.4.2 Product Inhibition 823.4.3 The Haldane Relationship 84References 854 Enzyme Inhibition and RelatedMechanisms 874.1 Unspecific and Irreversible Inhibition 874.1.1 Unspecific Inhibition 874.1.2 Irreversible Inhibition 884.1.2.1 General Features of Irreversible Inhibition 884.1.2.2 Suicide Substrates 904.1.2.3 Transition-State Analogs 914.1.2.4 Analysis of Irreversible Inhibition 924.2 Reversible Inhibition 944.2.1 General Rate Equation 944.2.1.1 Noncompetitive Inhibition and Graphic Representation of Inhibition Data 974.2.1.2 Competitive Inhibition 1024.2.1.3 Uncompetitive Inhibition 1064.2.2 Partial Inhibitions 1084.2.2.1 Partially Noncompetitive Inhibition 1084.2.2.2 Partially Uncompetitive Inhibition 1104.2.2.3 Partially Competitive Inhibition 1114.2.3 Noncompetitive and Uncompetitive Product Inhibition 1134.2.4 Substrate Inhibition 1144.3 Enzyme Reactions with Two Competing Substrates 1164.4 Different Enzymes Catalyzing the Same Reaction 118References 1195 Multi-Substrate Reactions 1215.1 Nomenclature 1215.2 Multi-Substrate Mechanisms 1225.2.1 Random Mechanism 1225.2.2 Ordered Mechanism 1275.2.3 Ping-Pong Mechanism 1295.2.4 Product Inhibition in Multi-Substrate Reactions 1315.2.5 Haldane Relationships in Multi-Substrate Reactions 1325.2.6 Mechanisms with MoreThan Two Substrates 1335.2.7 Other Nomenclatures for Multi-Substrate Reactions 1345.3 Derivation of Rate Equations of Complex Enzyme Mechanisms 1355.3.1 King–Altmann Method 1355.3.2 Simplified Derivations Applying GraphTheory 1405.3.3 Combination of Equilibrium and Steady-State Approach 141References 1436 pH and Temperature Dependence of Enzymes 1456.1 pH Optimum and Determination of pK Values 1456.2 pH Stability 1476.3 Temperature Dependence 148References 1527 Special EnzymeMechanisms 1537.1 Kinetic Treatment of Allosteric Enzymes 1537.2 Hysteretic Enzymes 1547.3 Kinetic Cooperativity, the Slow Transition Model 1557.4 Ribozymes 1567.5 Enzymes Reacting with Polymeric Substrates 159References 1608 Enzymes Bound to Artificial Matrices and to Membranes 1638.1 Immobilized Enzymes 1638.1.1 Kinetics of Immobilized Enzymes 1638.1.2 External Diffusion Limitation 1658.1.3 Internal Diffusion Limitation 1668.1.4 Inhibition of Immobilized Enzymes 1688.1.5 pH and Temperature Behavior of Immobilized Enzymes 1698.2 Enzyme Reactions at the Membrane 1698.2.1 Transport Processes 1698.2.2 Enzyme Reactions at Membrane Interfaces 172References 1759 Isotope Exchange and Isotope Effects 1779.1 Isotope Exchange 1779.1.1 Isotope Exchange Kinetics 1779.2 Isotope Effects 1819.2.1 Primary Kinetic Isotope Effect 1819.2.2 Influence of the Kinetic Isotope Effect on V and Km 1829.2.3 Other Isotope Effects 183References 18410 Related Subject Areas 18510.1 Relationship between Enzyme Kinetics and Pharmacokinetics 18510.2 Application of StatisticalMethods in Enzyme Kinetics 18910.2.1 General Remarks 18910.2.2 Statistical Terms Used in Enzyme Kinetics 191References 19311 Methods for the Study of Multiple Equilibria 19511.1 General Aspects 19511.2 Equilibrium Dialysis as an Example for the Performance of Binding Measurements 19711.2.1 Principle of Equilibrium Dialysis 19711.2.2 Control Experiments and Sources of Error 20011.2.2.1 Dialysis Time 20011.2.2.2 Concentration and Activity of the Macromolecule 20011.2.2.3 Concentration of the Ligand 20111.2.2.4 Donnan Effect 20211.2.3 Continuous Equilibrium Dialysis 20311.3 Ultrafiltration 20611.4 Gel Filtration 20811.4.1 Batch Method 20811.4.2 The Method of Hummel and Dreyer 20911.4.3 Other Gel FiltrationMethods 21011.5 Ultracentrifugation 21211.5.1 Fixed-Angle UltracentrifugationMethods 21211.5.2 Sucrose-Gradient Centrifugation 21511.6 Surface Plasmon Resonance 218References 22012 Manometric, Electrochemical, and Calorimetric Methods 22312.1 Warburg’s Manometric Apparatus 22312.2 Electrochemical Methods 22412.2.1 The Oxygen Electrode 22412.2.2 The CO2 Electrode 22612.2.3 Potentiometry, Redox Potentials 22712.2.4 The pH-Stat 22712.2.5 Polarography 22912.3 Calorimetry 230References 23213 Absorption and Fluorescence Spectroscopy 23513.1 General Aspects 23513.2 Absorption Spectroscopy 23713.2.1 The Lambert–Beer Law 23713.2.2 Spectral Properties of Enzymes and Ligands 23813.2.3 Structure of Spectrophotometers 24113.2.4 Double-Beam Spectrophotometer 24513.2.5 Difference Spectroscopy 24613.2.6 The Dual-Wavelength Spectrophotometer 24913.3 Photochemical Action Spectra 25013.4 Bioluminescence 25113.5 Fluorescence Spectroscopy 25113.5.1 Quantum Yield 25113.5.2 Structure of Spectrofluorometers 25213.5.3 Perturbations of Fluorescence Measurements 25413.5.4 Fluorescent Compounds (Fluorophores) 25513.5.5 Radiationless Energy Transfer 26013.5.6 Fluorescence Polarization 26213.5.7 Pulse Fluorometry 26313.5.8 Fluorescence Correlation Spectroscopy 265References 26514 Other Spectroscopic Methods 26914.1 Circular Dichroism and Optical Rotation Dispersion 26914.2 Infrared and Raman Spectroscopy 27414.2.1 IR Spectroscopy 27414.2.2 Raman Spectroscopy 27514.2.3 Applications 27514.3 Nuclear Magnetic Resonance Spectroscopy 27614.4 Electron Paramagnetic Resonance Spectroscopy 279References 28115 Methods to Measure Fast Reactions 28315.1 General Aspects 28315.2 Flow Methods 28415.2.1 The Continuous-Flow Method 28415.2.2 The Stopped-Flow Method 28715.2.3 Measurement of Enzyme Reactions by Flow Methods 29115.2.4 Determination of the Dead Time 29315.3 Relaxation Methods 29415.3.1 The Temperature-Jump Method 29415.3.2 The Pressure-Jump Method 29715.3.3 The Electric Field Method 29915.4 Flash Photolysis, Pico- and Femtosecond Spectroscopy 30015.5 Evaluation of Rapid Kinetic Reactions (Transient Kinetics) 302References 305Index 307