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Enzymes are Proteins with Defined 3D Structures

Enzymes are Proteins with Defined 3D Structures. Ribonuclease A. a -chymotrypsin. 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC). Active site:. Enzyme Catalysis: What Enzymes Can & Can’t Do. Acid-catalysed reaction. Enzyme-catalysed reaction. Types of Enzyme Assay.

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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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