Enzymes

Cover Contents 1. Introduction to Enzymes Introduction Meaning and definition History General properties of enzymes 1. Higher Reaction Rates 2. Milder Reaction Condition 3. Greater Reaction Specificity 4. Capacity for Regulations 5. Presence of Species Specificity 6. Variations in Activity and Ability 7. Substrate Specificity 8. Activation and Inhibition Sources of enzymes Characteristics of enzymes Questions 2. Enzyme Nomenclature and Classification Nomenclature Classification Major Classes 1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases (Synthetases) Key to Numbering and Classification of Enzymes 1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases Examples Questions 3. Enzyme Extraction and Purification Importance of Enzyme Purification General Procedure Extraction Enzyme Histochemistry Use of Centrifugation Extraction of Soluble Enzymes Extraction of Membrane-Bound Enzymes Nature of the Extraction Medium Purification of Enzymes Preliminary Procedure Fractionation Methods Partition Chromatography Electrophoresis Cation Exchange Chromatography Gel Filteration Affinity Chromatography Hplc Technology Lyophilization Determination of Molecular Weight of Enzymes Examples of Purification Procedure Adenylate Kinase from Pig Muscles Ribulose bisphosphate carboxylase from spinach RNA Polymerase from E.Coli Arom multi enzyme protein from Neurospora Glutathione reductase from E.Coli Adenylate Cyclase Chymosin Questions 4. Enzyme Assay Factors controlling in assays 1. Salt Concentration 2. Temperature 3. pH 4. Substrate Saturation 5. Level of Crowding Discontinous Assay Continous Assay Coupled Assay Enzyme Assay Techniques Visible and ultraviolet spectrophotometer methods Spectrofluorimetric method Luminescence method Radioisotope method Questions 5. Types of Enzyme Introduction Coenzymes Characteristics of coenzymes Isoenzymes Lactate dehydrogenase enzyme (LDH) Monomeric Enzymes Proteases (proteolytic enzymes) Oligomeric Enzymes Lactate Dehydrogenase Lactose Synthase Tryptophan Synthase Pyruvate Dehydrogenase Metalloenzymes and Metal-activated Enzymes Metalloenzymes Metal Activation Extremozymes Abzymes Ribozymes Synzymes Bi and Polyfunctional Enzyme Questions 6. Enzyme Catalysis Mechanisms in Organic Chemistry Mechanisms of Enzyme Catalysis Acid–Base Catalysis Strain Distortion and Conformational Change Catalysis by Approximation (Entrophic Contribution) Covalent Catalysis(Nucleophilic vs Electrophilic catalysis) Coenzymes in Enzyme-Catalyzed Reactions Questions 7. Enzyme Specificity Group Specificity Absolute Specificity Stereochemical Specificity The Active Sites Fischer’s Lock and Key Hypothesis Koshland Induced Fit Hypothesis Non-Productive Binding Transition State Stabilization Cryoenzymology Questions 8. Enzyme Kinetics Goals of Enzyme Kinetics Chemical Kinetics Reaction Order Classification of Chemical Reactions by Kinetic Order Kinetics of Bisubstrate Enzyme Reactions The Michaelis–Menten Equation The Lineweaver–Burk Plot The Eadie - Hofstee and Hans Plot Kinetics of Multisubstrate Enzyme-Catalyzed Reactions Ping-Pong Bi-Bi Mechanism Random-Order Mechanism Compulsory-Order Mechanism Steady-State Kinetics Plots For Mechanisms Which Follow the General Rate Equation Allosteric Enzymes Monod–Wyman Changeux (MWC) Model MWC Model and Allosteric Regulation Questions 9. Enzyme Inhibition Reversible Inhibition Competitive Inhibition Uncompetitive Inhibition Non-competitive Inhibition and Mixed Inhibition Partial Inhibition Substrate Inhibition Allosteric Inhibiton Irreversible Inhibition Questions 10. Enzyme Cofactors Mode of Action Cofactors Acting as Carriers Redox Carriers Phosphate Carriers CO2 Carrier Amino Group Carriers Acyl Group Carriers Carrier of One Carbon Group Sulphate Carrier Aldehyde Carrier Questions 11. Enzyme Biology Introduction Intracelluar Localization of Enzymes Histochemical Methods Separation of Particulate Fraction Questions 12. Enzyme Engineering Definition Objectives Principle of Enzyme Engineering Procedure in Enzyme Engineering Example Protein Engineering Addition of Disulphide Bond Changing Asparagine to other Aminoacids Reduction of Number of Free Sulphydryl Residues Increase of Enzymatic Activity Modification of Enzyme Specificity Secretion of Cloned Proteins Large Scale Fermentation Principles of Microbial Growth Bioreactors Harvesting of Microbial Cells Disruption of Microbial Cells Downstream Processing Questions 13. Immobilization of Enzyme Advantages Methods of Enzyme Immobilization Adsorption Covalent Bonding Entrapment Crosslinking Encapsulation Applications of Immobilized Enzyme Immobilized Enzyme Kinetics Heterogenous Concentrations Concentration Gradients and Reaction Rates in Solid Catalyst Effect of External Mass Transfer Resistance Diffusion Effects in Surface-Bound Enzymes on Non-Porous Support Materials Design of Immobilized Enzyme Reactors Batch Reactors Continuous Flow Reactors Packed Bed Reactor Fluidized-Bed Reactors Membrane Reactors Questions 14. Biosensors Principle General Features Glucose Electrode Types of Biosensors Calorimetric Biosensors Amperometric Biosensors Optical Biosensors Acoustic Wave Biosensor Potentiometric Biosensors Whole Cell Biosensors (Microbial Biosensors) Applications of Biosensors Questions 15. Clinical Enzymology Amylase Transaminases Clinical Significance Alkaline Phosphatase (ALP) Clinical Significance Acid Phosphatase (ACP) Clinical Significance Lactate Dehydrogenase (LD) Clinical Significance Creatine Kinase (CK) Clinical Significance Gamma-Glutamyl Transferase (GGT) Clinical Significance Cholinesterases Clinical Significance Questions Clinical Significance 16 Applications in Enzymes Applications in Medicine Applications in Medicine Assay of Plasma Enzymes Therapeutic Uses Application In Industry In Food Industry Baking of Bread Enzymes and Recombinant DNA Technology In Dairy Industry Cheese Making In Starch Industry In Detergent Industry In Leather Industry Wool Industry Production Syrups In Sugar Industry In Brewing Industry Biotechnological Applications of Enzymes Analytical Uses Principles of Enzymatic Analysis Questions