Enzymes are Proteins with Defined 3D Structures

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Enzyme Kinetics C483 Spring 2013.

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Presentation transcript:

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 1 Unit = activity required to convert 1 µmole S to P per minute

Fluorescence Resonance Energy Transfer Assay for 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).

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

Michaelis-Menten Model for Enzyme Kinetics Kinetic Model

Graphical Determination of Km & kcat

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]

Enzyme Inhibition - Reversible

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

Inhibition by 2’-deoxyadenosine analogue IC50 = 100 µM

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

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) + A2 exp (-k2t) Triple Exponential Mode A= A0 + A1 exp (-k1t) + A2 exp (-k2t) + A3 exp (-k3t)

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

Analysis of His263Ala Mutant Kinetic Parameters pH=8.0 KM (μM) kcat (s-1) kcat/ KM (M-1s-1) WT 6.8 28 4.1 x 106 H263A 5.5 0.0029 5.3 x 102 Pre-steady state Kinetic Parameters 0.037 -55.4 0.223 -146 H263A 18 -117 144 -131 Wild type. 270nm (dienol P) 0.040 78.5 0.34 96.6 153.2 145.6 317nm (dienol S) k2 (s-1) A2 (×103) k1 (s-1) A1 (×103) pH=7.0 0.34s-1 0.04s-1 H263 is involved in both ketonization and C-C cleavage !

Analysis of Ser110Ala Mutant Kinetic Parameters pH=8.0 KM (μM) kcat (s-1) kcat/ KM (M-1s-1) WT 6.8 28 4.1 x 106 S110A 18.5 0.0054 2.9 x 102 Pre-steady state Kinetic 20ms 317nm 200ms 317nm 200s 317nm 140s-1 0.02s-1 3.1s-1