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Enzymes are Proteins with Defined 3D Structures

Dive deep into enzyme proteins, active sites, catalysis models, and inhibition mechanisms. Explore kinetic parameters and different enzyme assays. Learn about reversible and irreversible inhibition, and analysis of enzyme mutants.

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Enzymes are Proteins with Defined 3D Structures

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  1. Enzymes are Proteins with Defined 3D Structures Ribonuclease A a-chymotrypsin 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) Active site:

  2. Enzyme Catalysis: What Enzymes Can & Can’t Do Acid-catalysed reaction Enzyme-catalysed reaction

  3. Types of Enzyme Assay 1 Unit = activity required to convert 1 µmole S to P per minute

  4. Fluorescence Resonance Energy Transfer Assay for MurG MurG N-dansyl lipid I Ex 290 nm 340 nm 0.2 M Tris pH 7.5, 10 mM MgCl2, 0.2% CHAPS 2.7 µM Fl UDPGlcNAc, 3.0 µM dansyl lipid I + 3.0 µg E. coli MurG Em 500 nm J.J. Li and T.D.H. Bugg,Chem. Commun., 182-183 (2004).

  5. Enzyme Purification Preparation of Cell Extract SDS-PAGE gel Purification Table

  6. Michaelis-Menten Model for Enzyme Kinetics Kinetic Model

  7. Graphical Determination of Km & kcat

  8. What do Km & kcat really mean? kcat - turnover number 1st order rate constant (units s-1) for turnover at high [S] Km - Michaelis constant Measure of affinity of Substrate binding BUT not the same as Kd! kcat/Km - catalytic efficiency 2nd order rate constant (units M-1 s-1) for turnover at low [S]

  9. Enzyme Inhibition - Reversible

  10. Transition State Analogues for Ligase MurM Inhibitor design: mimic tetrahedral transition state: Transition state Phosphonate analogue

  11. Inhibition by 2’-deoxyadenosine analogue IC50 = 100 µM

  12. Enzyme Inhibition - Irreversible Inhibition e.g. serine protease inhibitor DFP

  13. Pre-Steady State Kinetics —— Application to C-C Hydrolase MhpC Data Simulation • Single Exponential Mode • A = A0 +A1 exp (-k1t) • Double Exponential Mode • A= A0 + A1 exp (-k1t) + A2exp (-k2t) • Triple Exponential Mode • A= A0 + A1 exp (-k1t) + A2exp (-k2t) + A3exp (-k3t)

  14. Fit with single exponential (1 step) Fit with double exponential (2 step)

  15. pH=7.0 A1 (×103) k1 (s-1) A2 (×103) k2 (s-1) 317nm (dienol S) Wild type. 145.6 153.2 H263A 96.6 0.34 78.5 0.040 270nm (dienol P) Wild type. -131 144 -117 18 H263A -146 0.223 -55.4 0.037 Analysis of His263Ala Mutant • Kinetic Parameters • Pre-steady state Kinetic Parameters 0.04s-1 0.34s-1 H263 is involved in both ketonization and C-C cleavage !

  16. 20ms 317nm 200ms 317nm 200s 317nm 140s-1 0.02s-1 3.1s-1 Analysis of Ser110Ala Mutant • Kinetic Parameters • Pre-steady state Kinetic

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