1. Enzymes are Proteins with Defined 3D Structures Ribonuclease A 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC)  -chymotrypsin 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. MurG N-dansyl lipid I Ex 290 nm 340 nm Em 500 nm Fluorescence Resonance Energy Transfer Assay for MurG 0.2 M Tris pH 7.5, 10 mM MgCl 2, 0.2% CHAPS 2.7 µM Fl UDPGlcNAc, 3.0 µM dansyl lipid I + 3.0 µg E. coli MurG J.J. Li and T.D.H. Bugg,Chem. Commun., 182-183 (2004).

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

6. Michaelis-Menten Model for Enzyme Kinetics Kinetic Model

7. Graphical Determination of K m & k cat

8. What do K m & k cat really mean? k cat - turnover number 1st order rate constant (units s -1 ) for turnover at high [S] K m - Michaelis constant Measure of affinity of Substrate binding BUT not the same as K d ! k cat /K m - 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 statePhosphonate analogue

11. Inhibition by 2’-deoxyadenosine analogue IC 50 = 100 µM

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

13. Data Simulation  Single Exponential Mode A = A 0 +A 1 exp (-k 1 t) A = A 0 +A 1 exp (-k 1 t)  Double Exponential Mode A= A 0 + A 1 exp (-k 1 t) + A 2 exp (-k 2 t) A= A 0 + A 1 exp (-k 1 t) + A 2 exp (-k 2 t)  Triple Exponential Mode A= A 0 + A 1 exp (-k 1 t) + A 2 exp (-k 2 t) + A 3 exp (-k 3 t) A= A 0 + A 1 exp (-k 1 t) + A 2 exp (-k 2 t) + A 3 exp (-k 3 t) Data Simulation  Single Exponential Mode A = A 0 +A 1 exp (-k 1 t) A = A 0 +A 1 exp (-k 1 t)  Double Exponential Mode A= A 0 + A 1 exp (-k 1 t) + A 2 exp (-k 2 t) A= A 0 + A 1 exp (-k 1 t) + A 2 exp (-k 2 t)  Triple Exponential Mode A= A 0 + A 1 exp (-k 1 t) + A 2 exp (-k 2 t) + A 3 exp (-k 3 t) A= A 0 + A 1 exp (-k 1 t) + A 2 exp (-k 2 t) + A 3 exp (-k 3 t) Pre-Steady State Kinetics —— Application to C-C Hydrolase MhpC

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

15. 0.037-55.40.223-146H263A 18-117144-131Wild type. 270nm (dienol P) 0.04078.5 0.34 96.6H263A 153.2145.6Wild type. 317nm (dienol S) k 2 (s -1 ) A 2 (×10 3 ) k 1 (s -1 ) A 1 (×10 3 ) pH=7.0 0.34s -1 0.04s -1 H263 is involved in both ketonization and C-C cleavage ! Analysis of His263Ala Mutant pH=8.0 K M (μM) k cat (s -1 ) k cat / K M (M -1 s -1 ) WT6.8284.1 x 10 6 H263A5.50.00295.3 x 10 2 Kinetic Parameters Kinetic Parameters Pre-steady state Kinetic Parameters Pre-steady state Kinetic Parameters

16. 20ms 317nm 200ms 317nm 200s 317nm Analysis of Ser110Ala Mutant pH=8.0 K M (μM) k cat (s -1 ) k cat / K M (M -1 s -1 ) WT6.8284.1 x 10 6 S110A18.50.00542.9 x 10 2 Kinetic Parameters Kinetic Parameters Pre-steady state Kinetic Pre-steady state Kinetic 140s -1 0.02s -1 3.1s -1