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Enzymology Dr. Nasir Jalal ASAB, National University of Sciences and Technology Slide 2 Course content Introduction and history of enzymes Historical aspects Discovery of enzymes Chemistry of enzymes Function and importance Enzymes in biotechnology Characteristics and properties Catalytic power and specificity Enzymes as catalysts Enzyme - substrate interactions Lock & key model Induced fit model Transition state model Quantum tunnelling model Enzymes as proteins Non-protein cofactors Metal ions Organic cofactors Nomenclature / Classification and Activity Measurements Oxidoreductase-dehydrogenase Transferase Hydrolase Lyase Isomerase Ligase Activity measurements Enzyme Purification and Assay Initial velocity measurements Assay types Enzyme units of activity Turnover number and properties Purification and assessment Methods for measurement Enzyme kinetics Michaelis-Menten Kinetics Introduction Assumptions Derivation Description of v o versus [S] Michaelis constant (K M ) Slide 3 Course content Specificity/Substrate constant (SpC) Graphical Analysis of Kinetic Data, pH and Temp Dependence Graphical Analysis Lineweaver-Burk Analysis Hanes-Woolf Analysis Eadie-Hofstee Analysis Direct Linear Plot (Eisenthal/Cornish-Bowden Plot) Nonlinear Curve Fitting pH-dependence of Michaelis-Menten Enzymes Temperature-Dependence of Enzyme Reactions Single Molecule Enzymology ATP Synthase ATP Synthase with Tethered Actin Myosin-V Kinesin motor attached to a fluorescent bead Single Molecule Studies of Cholesterol Oxidase -galactosidase: a model Michaelis-Menten enzyme? Enzyme inhibition and kinetics Classification of inhibitors Reversible, Irreversible, Iodoacetamide, DIFP Classification of Reversible Inhibitors Competitive, Uncompetitive, Noncompetitive, Substrate Multi-substrate Reactions and Substrate Binding Analysis Substrate Binding Analysis Single Binding Site Model Binding Data Plots Direct Plot Reciprocal Plot Scatchard Plot Determination of Enzyme-Substrate Dissociation Constants Kinetics Equilibrium Dialysis Equilibrium Gel Filtration Ultracentrifugation Spectroscopic Methods Mechanism of enzyme catalysis Engineering More Stable Enzymes Incorporation of Non-natural Amino Acids into Enzymes Protein Engineering by Combinatorial Methods DNA Shuffling Slide 4 My office hours Thursdays 9:00-11:00 Fridays 2:00-4:00 Nasir.Jalal@doctor.com Slide 5 Introduction to enzymology Enzymes are Biomolecules that catalyze, increase the rates of chemical reactions by 10 15 to 10 17 fold. Almost all enzymes are proteins. In enzymic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, the products. Living systems use enzymes to accelerate and control the rates of vitally important biochemical reactions. Slide 6 Brief history Earliest known use of enzymes comes from the Egyptian civilization which used yeast for fermentation and called the product Boza. HISTORY of Enzymes As early as the late 1700s and early 1800s, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known. However, the mechanism by which this occurred had not been identified. Slide 7