FUNDAMENTALS OF ENZYMOLOGY

Transcription

1 FUNDAMENTALS OF ENZYMOLOGY Meenakshi Meena Deepak Chauhan Aavishkar Publishers, Distributors Jaipur (Raj.) India

2 Contents Preface v 1. INTRODUCTION 1 Historical Aspects 1 Enzymes and Life Processes 2 Early Enzyme Discoveries 3 Chemical Nature of Enzymes 3 Specificity of Enzymes 4 Naming and Classification 5 General Principles 5 Recommended and Systematic Names., 8 Scheme of Classification and Numbering of Enzyme-Catalysed Reactions 9 Class 1. Oxidoreductases 9 Class 2. Transferases 10 -Class 3. Hydrolases 11 Class 4. Lyases 11 Class 5. Isomerases 12 Class 6. Ligases 12 Rules for Classification and Nomenclature 13

3 viii -(a) General Rules for Systematic Names and Guidelines for Recommended Names 13 -(b) Rules and Guidelines for Particular Classes of "Enzymes PURIFICATION OF ENZYMES 23 Sources of Enzymes 23 Screening for Novel Enzymes 26 Media for Enzyme Production 28 Preparation of Enzymes 29 Centrifugation 31 Aqueous Biphasic Systems 35 -Cell Breakage 39 Ultrasonic Cell Disruption 40 High Pressure Homogenisers 41 -Use of Bead Mills 43 Use of Freeze-presses 44 Use of Lytic Methods 44 Preparation of Enzymes from Clarified Solution 45 Nucleic Acid Removal 46 Concentration by Precipitation 46 -Heat Treatment 47 Chromatography 47 Ion-exchange Chromatography 48 Affinity Chromatography 49 Gel Exclusion Chromatography 51 -Preparation of Enzymes for Sale 51 -Safety and Regulatory Aspects of Enzyme use CATALYSIS 56 Methods of Catalysis-1 56 General Acid/Base Catalysis 56 -Metal Ion (or Electrostatic) Catalysis 57 -Covalent or Nucleophilic Catalysis 59 Intramolecular Catalysis 62 Transition State Stabilization 67 Abyzmes-Antibody Catalysis 68 Ribozymes 69

4 ix -Catalysis Carboxypeptidase 70 Lysozyme 71 Chymotrypsin 74 Proteases Mechanisms 80 Proteasome 83 Other Types of Enzymes New Ideas on Enzyme Catalysis Catalysis-Ill 86 Ribozymes THE MECHANISM OF ENZYME CATALYSIS 95 Simple Kinetics of Enzyme Action 97 -Effect of ph and Ionic Strength 102 Effect of Temperature and Pressure 107 Reversible Reactions HI Enzyme Inhibition 118 Competitive Inhibition 120 Uncompetitive Inhibition 122 Noncompetitive Inhibition 124 Determination of Vmax and Km 124 Regulation of Enzyme Activity 128 Allosteric Enzymes IMMOBILIZATION OF ENZYMES 134 The Economic Argument for Immobilization 134 Methods of Immobilization 135 Kinetics of Immobilised Enzymes 136 Cyanogen Bromide 141 -Ethyl Chloroformate 142 Carbodiimide 142 -Glutaraldehyde arninopropyltriefhoxysilane 143 Effect of Solute Partition on the Kinetics of Immobilised Enzymes 146 Effects of Solute Diffusion on the Kinetics of Immobilised Enzymes 152 Analysis of Diffusional Effects in Porous Supports 170 Enzyme Reactors 181

5 X Membrane Reactors 184 Continuous Flow Reactors 185 Packed Bed Reactors 186 Continuous Flow Stirred Tank Reactors 188 -Fluidised Bed Reactors 193 Immobilised-enzyme Processes 195 -High-fructose Corn Syrups (HFCS) 195 Use of Immobilised Raffinase 200 Use of Immobilised Invertase 200 Production of Amino Acids 201 Use of Immobilised Lactase 202 -Production of Antibiotics 203 Preparation of Acrylamide BIOSENSORS 205 Calorimetric Biosensors 208 Potentiometric Biosensors 211 Amperometric Optical Biosensors 215 Biosensors 219 -Piezo-electric Biosensors 220 Immunosensors, RECENT ADVANCES IN ENZYME TECHNOLOGY 223 Enzymic Reactions in Biphasic Liquid Systems 223 -The Stabilisation of Enzymes in Biphasic Aqueous-organic Systems 224 Equilibria in Biphasic Aqueous-organic Systems 227 -Enzyme Kinetics in Biphasic Aqueous-organic Systems 235 -Use of Aqueous Two-phase Systems 236 -Practical Examples of the use of Enzymes 'In Reverse' 237 Glycosidases Used in Synthetic Reactions 241 Interesterification of Lipids 242 The Large-scale Use of Enzymes in Solution 243 The Use of Enzymes in Detergents 243 Applications of Proteases in the Food Industry 247 The Use of Proteases in the Leather and Wool Industries 250 The Use of Enzymes in Starch Hydrolysis 251 Production of Glucose Syrup ; 254

6 xi Production of Syrups Containing Maltose 256 Enzymes in the Sucrose Industry 258 Glucose from Cellulose 259 The use of Lactases in the Dairy Industry 261 -Enzymes in the Fruit Juice, Wine, Brewing and Distilling Industries 262 -Glucose Oxidase and Catalase in the Food Industry 264 Medical Applications of Enzymes FUTURE PROSPECTS FOR ENZYME TECHNOLOGY 267 Whither Enzyme Technology 267 Use of 'Unnatural' Substrates 268 Enzyme Engineering 269 -Artificial Enzymes 273 Coenzyme-regenerating Systems 274 The use of Enzymes in Analysis BIOENERGETICS 278 Thermodynamics and its Application 279 -The Basics of Usable Energy.'. 279 Heat and Efficiency 280 -Brief History & Orientation 281 Oxygen Electrodes 284 Oxygen Electrode (Oxygraph) Recordings 285 Respiratory Chain Components 286 Uncouplers. and Inhibitors 289 Sub-mitochondrial Particles 293 -The High-Energy Pool 294 -The FOFl-ATPase, 297 Mitochondrial Transport Systems 303 Origin & Evolution of Mitochondria 308 Biogenesis of Mitochondria 310 Mitochondrial Diseases 312 Chemical Structures PHOTOSYNTHESIS 319 -Plant Photosynthesis 320 Photosynthesis in Algae and Bacteria 321 Molecular Production 321

7 - -OEC xii Light to Chemical Energy 321 -Z Scheme 323 Water Photolysis 323 Oxygen and Photosynthesis 323 Bacterial Variations 324 Discovery 324 Bioenergetics of Photosynthesis 325 Factors Affecting Photosynthesis 326 -Light Intensity (Irradiance), Wavelength and Temperature 326 Carbon Dioxide 327 Corollary Factors 328 -Chlorophyll 328 -Chemical Structure 329 Evidence for Chlorophyll 331 Oxidation/Phosphorylation 331 -The Light Reaction 332 -Photosystem II 334 -In Summary, for PSII in Plants 336 Complex 336 -Plant Protection 336 Production of Hydrogen : Hydrogenases 338 -Calvin Cycle : Photosynthetic Carbon Reactions 339 -C3 vs C4 plants 341 Microbial Metabolism 344 -ATP NAD 346 -ATP Synthesis 347 -Heterotrophic Types of Metabolism 349 Fermentation 349 -The Embden-Meyerhof Pathway 352 Respiration 355 Anaerobic Respiration 356 Lithotrophic Types of Metabolism 358 Phototrophic Metabolism 360 -Autotrophic C02 Fixation 367

8 xiii Biosynthesis METALLOENZYMES 371 Introduction 371 Structure and Overview General Functions of Metalloenzym.es 373 Hemoglobins 373 Cytochromes 373 Phosphotransferase 373 Alcohol Dehydrogenase 373 Arginase Ferredoxin 373 Cytochrome Oxidase 373 Carboxypeptidase A 373 -CPA Binding of Substrate 373 CPA Hydrolyzes Oligopeptide 374 Regulation and Control 374 Metalloenzyme Inhibition 374 Regulation of Metalloenzymes Through Diet 375 Theoretical Study of Metalloenzymes 376 Structural Characteristic of Zinc Enzymes 378 Classification of Zinc Sites 378 Mononuclear Zinc Environment 384 Catalytic Mechanisms of Zinc Enzymes 386 Zinc Proteases 386 Reverse Protonation Mechanism 389 Dizinc Proteases 393 Other Zinc Enzymes 396 Bibliography 404 Index 418