Creatinine factory in semi-automatic chemistry

1. Enzymes are Proteins with Defined 3D Structures
Ribonuclease A
2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC)
a-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 MgCl2, 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 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. 0.0 0.2 0.4 0.6 0.8 1.0
0
20
40
60
80
100
MurM
activity
(%)
Inhibitor Conc (mM)
Inhibition by 2’-deoxyadenosine analogue
IC50 = 100 µM

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

13. 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)
Pre-Steady State Kinetics —— Application to C-C Hydrolase MhpC
CO2
O
O2C OH
CO2
O
O2C O
O2C OH CO2
CO2
-
-
-
-
+
Low pH A317 High pH A394
MhpC
-
+
-
-
A270

14. 0
1
0.4
0.6
0.8
1
1.2
1.4
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Association Time (s) -2
× 10
Position
(Arc
s)
× 10-2
-4
-3
-2
-1
0
1
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Association Time (s) -2
× 10
Error
(Arc
s)
× 10-1
0
1
2
0.5
1
1.5
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Association Time (s) -2
× 10
Position
(Arc
s)
× 10-2
-5
-2.5
0
2.5
5
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Association Time (s) -2
× 10
Error
(Arc
s)
Fit with single exponential (1 step) Fit with double exponential (2 step)

15. 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
H263A
153.2
145.6
Wild type.
317nm
(dienol S)
k2 (s-1)
A2 (×103)
k1 (s-1)
A1 (×103)
pH=7.0
E + RFP E.RFP E.RFP E.succ E + succinate
150s 18 s
-1 -1
k
0.34s-1 0.04s-1
H263 is involved in both ketonization and C-C cleavage !
Analysis of His263Ala Mutant
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
 Kinetic Parameters
 Pre-steady state Kinetic Parameters

16. 20ms 317nm 200ms 317nm 200s 317nm
Analysis of Ser110Ala Mutant
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
 Kinetic Parameters
 Pre-steady state Kinetic
E + RFP E.RFP E.RFP E.succ E + succinate
150s 18 s
-1 -1
k
E.RFP*
140s-1 0.02s-1
3.1s-1