Details

Enzyme Kinetics

Principles and Methods
3rd Enlarged and Improved Edition

von: Hans Bisswanger
133,99 €
Verlag:	Wiley-VCH
Format:	PDF
Veröffentl.:	22.06.2017
ISBN/EAN:	9783527806478
Sprache:	englisch
Anzahl Seiten:	336

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Beschreibungen

Titelbeschreibung

Now in full color for a more intuitive learning experience, this new edition of the long-selling reference also features a number of new developments in methodology and the application of enzyme kinetics. Starting with a description of ligand binding equilibria, the experienced author goes on to discuss simple and complex enzyme reactions in kinetic terms. Special cases such as membrane-bound and immobilized enzymes are considered, as is the influence of external conditions, such as temperature and pH value. The final part of the book then covers a range of widely used measurement methods and compares their performance and scope of application. With its unique mix of theory and practical advice, this is an invaluable aid for teaching as well as for experimental work.

Inhaltsverzeichnis

Preface xi Symbols and Abbreviations xiii Introduction and Definitions xv 1 Multiple Equilibria, Principles, and Derivations 1 1.1 General Considerations 1 1.2 Diffusion 2 1.3 Modes of Ligand Binding 4 1.4 Interaction between Macromolecules and Ligands 6 1.4.1 Binding Constants 6 1.4.2 Binding to a Single Site 7 1.5 Binding to Identical Independent Sites 7 1.5.1 General Binding Equation 7 1.5.2 Graphic Representations of the Binding Equation 13 1.5.2.1 Direct and Linear Diagrams 13 1.5.2.2 Analysis of Binding Data from Spectroscopic Titrations 15 1.5.3 Binding of Different Ligands, Competition 18 1.5.4 Noncompetitive Binding 21 1.6 Binding to Nonidentical, Independent Sites 23 References 25 2 Cooperativity and Allosteric Enzymes 27 2.1 Binding to Interacting Sites 27 2.1.1 The Hill Equation 27 2.1.2 The Adair Equation 29 2.1.3 The Pauling Model 32 2.2 Allosteric Enzymes 32 2.2.1 The Symmetry or Concerted Model 33 2.2.2 The SequentialModel and Negative Cooperativity 38 2.2.3 Analysis of Cooperativity 42 2.2.4 Physiological Aspects of Cooperativity 44 2.2.5 Examples of Allosteric Enzymes 46 2.2.5.1 Hemoglobin 46 2.2.5.2 Aspartate Transcarbamoylase 48 2.2.5.3 Aspartokinase 49 2.2.5.4 Phosphofructokinase 50 2.2.5.5 Allosteric Regulation of the Glycogen Metabolism 50 2.2.5.6 Membrane-Bound Enzymes and Receptors 50 2.3 Binding to Nonidentical, Interacting Sites 51 References 52 3 FromReaction Order to the Michaelis–Menten Law: Fundamental Relationships of Enzyme Kinetics 55 3.1 Reaction Order 55 3.1.1 First-Order Reactions 56 3.1.2 Second-Order Reactions 57 3.1.3 Zero-Order Reactions 58 3.2 Steady-State Kinetics and the Michaelis–Menten Equation 58 3.2.1 Derivation of the Michaelis–Menten Equation 58 3.3 Analysis of Enzyme Kinetic Data 62 3.3.1 Graphic Representations of the Michaelis–Menten Equation 62 3.3.1.1 Direct and Semilogarithmic Representations 62 3.3.1.2 Direct Linear Plots 68 3.3.1.3 LinearizationMethods 70 3.3.2 Analysis of Progress Curves 72 3.3.2.1 Integrated Michaelis–Menten Equation 73 3.3.2.2 Determination of Reaction Rates 75 3.3.2.3 Graphic Methods for Rate Determination 77 3.3.2.4 Graphic Determination of True Initial Rates 79 3.4 Reversible Enzyme Reactions 80 3.4.1 Rate Equation for Reversible Enzyme Reactions 80 3.4.2 Product Inhibition 82 3.4.3 The Haldane Relationship 84 References 85 4 Enzyme Inhibition and RelatedMechanisms 87 4.1 Unspecific and Irreversible Inhibition 87 4.1.1 Unspecific Inhibition 87 4.1.2 Irreversible Inhibition 88 4.1.2.1 General Features of Irreversible Inhibition 88 4.1.2.2 Suicide Substrates 90 4.1.2.3 Transition-State Analogs 91 4.1.2.4 Analysis of Irreversible Inhibition 92 4.2 Reversible Inhibition 94 4.2.1 General Rate Equation 94 4.2.1.1 Noncompetitive Inhibition and Graphic Representation of Inhibition Data 97 4.2.1.2 Competitive Inhibition 102 4.2.1.3 Uncompetitive Inhibition 106 4.2.2 Partial Inhibitions 108 4.2.2.1 Partially Noncompetitive Inhibition 108 4.2.2.2 Partially Uncompetitive Inhibition 110 4.2.2.3 Partially Competitive Inhibition 111 4.2.3 Noncompetitive and Uncompetitive Product Inhibition 113 4.2.4 Substrate Inhibition 114 4.3 Enzyme Reactions with Two Competing Substrates 116 4.4 Different Enzymes Catalyzing the Same Reaction 118 References 119 5 Multi-Substrate Reactions 121 5.1 Nomenclature 121 5.2 Multi-Substrate Mechanisms 122 5.2.1 Random Mechanism 122 5.2.2 Ordered Mechanism 127 5.2.3 Ping-Pong Mechanism 129 5.2.4 Product Inhibition in Multi-Substrate Reactions 131 5.2.5 Haldane Relationships in Multi-Substrate Reactions 132 5.2.6 Mechanisms with MoreThan Two Substrates 133 5.2.7 Other Nomenclatures for Multi-Substrate Reactions 134 5.3 Derivation of Rate Equations of Complex Enzyme Mechanisms 135 5.3.1 King–Altmann Method 135 5.3.2 Simplified Derivations Applying GraphTheory 140 5.3.3 Combination of Equilibrium and Steady-State Approach 141 References 143 6 pH and Temperature Dependence of Enzymes 145 6.1 pH Optimum and Determination of pK Values 145 6.2 pH Stability 147 6.3 Temperature Dependence 148 References 152 7 Special EnzymeMechanisms 153 7.1 Kinetic Treatment of Allosteric Enzymes 153 7.2 Hysteretic Enzymes 154 7.3 Kinetic Cooperativity, the Slow Transition Model 155 7.4 Ribozymes 156 7.5 Enzymes Reacting with Polymeric Substrates 159 References 160 8 Enzymes Bound to Artificial Matrices and to Membranes 163 8.1 Immobilized Enzymes 163 8.1.1 Kinetics of Immobilized Enzymes 163 8.1.2 External Diffusion Limitation 165 8.1.3 Internal Diffusion Limitation 166 8.1.4 Inhibition of Immobilized Enzymes 168 8.1.5 pH and Temperature Behavior of Immobilized Enzymes 169 8.2 Enzyme Reactions at the Membrane 169 8.2.1 Transport Processes 169 8.2.2 Enzyme Reactions at Membrane Interfaces 172 References 175 9 Isotope Exchange and Isotope Effects 177 9.1 Isotope Exchange 177 9.1.1 Isotope Exchange Kinetics 177 9.2 Isotope Effects 181 9.2.1 Primary Kinetic Isotope Effect 181 9.2.2 Influence of the Kinetic Isotope Effect on V and Km 182 9.2.3 Other Isotope Effects 183 References 184 10 Related Subject Areas 185 10.1 Relationship between Enzyme Kinetics and Pharmacokinetics 185 10.2 Application of StatisticalMethods in Enzyme Kinetics 189 10.2.1 General Remarks 189 10.2.2 Statistical Terms Used in Enzyme Kinetics 191 References 193 11 Methods for the Study of Multiple Equilibria 195 11.1 General Aspects 195 11.2 Equilibrium Dialysis as an Example for the Performance of Binding Measurements 197 11.2.1 Principle of Equilibrium Dialysis 197 11.2.2 Control Experiments and Sources of Error 200 11.2.2.1 Dialysis Time 200 11.2.2.2 Concentration and Activity of the Macromolecule 200 11.2.2.3 Concentration of the Ligand 201 11.2.2.4 Donnan Effect 202 11.2.3 Continuous Equilibrium Dialysis 203 11.3 Ultrafiltration 206 11.4 Gel Filtration 208 11.4.1 Batch Method 208 11.4.2 The Method of Hummel and Dreyer 209 11.4.3 Other Gel FiltrationMethods 210 11.5 Ultracentrifugation 212 11.5.1 Fixed-Angle UltracentrifugationMethods 212 11.5.2 Sucrose-Gradient Centrifugation 215 11.6 Surface Plasmon Resonance 218 References 220 12 Manometric, Electrochemical, and Calorimetric Methods 223 12.1 Warburg’s Manometric Apparatus 223 12.2 Electrochemical Methods 224 12.2.1 The Oxygen Electrode 224 12.2.2 The CO2 Electrode 226 12.2.3 Potentiometry, Redox Potentials 227 12.2.4 The pH-Stat 227 12.2.5 Polarography 229 12.3 Calorimetry 230 References 232 13 Absorption and Fluorescence Spectroscopy 235 13.1 General Aspects 235 13.2 Absorption Spectroscopy 237 13.2.1 The Lambert–Beer Law 237 13.2.2 Spectral Properties of Enzymes and Ligands 238 13.2.3 Structure of Spectrophotometers 241 13.2.4 Double-Beam Spectrophotometer 245 13.2.5 Difference Spectroscopy 246 13.2.6 The Dual-Wavelength Spectrophotometer 249 13.3 Photochemical Action Spectra 250 13.4 Bioluminescence 251 13.5 Fluorescence Spectroscopy 251 13.5.1 Quantum Yield 251 13.5.2 Structure of Spectrofluorometers 252 13.5.3 Perturbations of Fluorescence Measurements 254 13.5.4 Fluorescent Compounds (Fluorophores) 255 13.5.5 Radiationless Energy Transfer 260 13.5.6 Fluorescence Polarization 262 13.5.7 Pulse Fluorometry 263 13.5.8 Fluorescence Correlation Spectroscopy 265 References 265 14 Other Spectroscopic Methods 269 14.1 Circular Dichroism and Optical Rotation Dispersion 269 14.2 Infrared and Raman Spectroscopy 274 14.2.1 IR Spectroscopy 274 14.2.2 Raman Spectroscopy 275 14.2.3 Applications 275 14.3 Nuclear Magnetic Resonance Spectroscopy 276 14.4 Electron Paramagnetic Resonance Spectroscopy 279 References 281 15 Methods to Measure Fast Reactions 283 15.1 General Aspects 283 15.2 Flow Methods 284 15.2.1 The Continuous-Flow Method 284 15.2.2 The Stopped-Flow Method 287 15.2.3 Measurement of Enzyme Reactions by Flow Methods 291 15.2.4 Determination of the Dead Time 293 15.3 Relaxation Methods 294 15.3.1 The Temperature-Jump Method 294 15.3.2 The Pressure-Jump Method 297 15.3.3 The Electric Field Method 299 15.4 Flash Photolysis, Pico- and Femtosecond Spectroscopy 300 15.5 Evaluation of Rapid Kinetic Reactions (Transient Kinetics) 302 References 305 Index 307

Autorenportrait

Hans Bisswanger was Professor at the Interfaculty Institute of Biochemistry at the University of Tubingen (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.