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
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., (2004).

5. Enzyme Purification Preparation of Cell Extract Purification Table
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) + A2 exp (-k2t)
Triple Exponential Mode
A= A0 + A1 exp (-k1t) + A2 exp (-k2t) + A3 exp (-k3t)

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

15. 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 !

16. 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