1. CHMI 2227E Biochemistry I Enzymes: Kinetics
CHMI E.R. Gauthier, Ph.D.

2. Enzymatic reactions Enzyme (each = 1 µmol)
Only concentrations we know  we’re the ones who set up the experiment!
Substrate (each = 1 µmol)
X min
Product
CHMI E.R. Gauthier, Ph.D.

3. Enzymatic reactions How do we measure enzyme activity?
1. Detection of the product(s):
pNA = para-nitroaniline  Absorbs at 405 nm
H3+N-CH-C-NH-CH-C-NH-CH-C-NH-CH-C-OH O
CH2
COO- CH
CH3
H3C
NO2
H2N
pNA (yellow)
H3+N-CH-C-NH-CH-C-NH-CH-C-NH-CH-C-NH-
DEVD-pNA
(uncolored)
DEVD
(uncoloured)
Caspase 3 (proteasehydrolase)
Measure increase
in A405nm
CHMI E.R. Gauthier, Ph.D.

4. Enzymatic reactions How do we measure enzyme activity?
2. Accumulation/utilisation of a co-factor:
NADH = absorbs strongly at 340 nm (e = 6.3 molL-1cm-1 )
NAD+ =does not absorb at 340 nm
Measure increase
in A340nm
Measure decrease
in A340nm
Lactate
dehydrogenase
CHMI E.R. Gauthier, Ph.D.

5. Enzymatic reactions How do we measure enzyme activity?
3. Coupled reactions:
Very useful when neither substrate/product/co-factor can be (easily) detected;
Glutaminase
+ NH4+
1st reaction
Measure increase
in A340nm
Glutamate
Dehydrogenase
+ NAD+
+ NADH +H+
2nd reaction
+ H2O
Detectable by HPLC
but not practical
CHMI E.R. Gauthier, Ph.D.

6. Enzymatic reactions [Product] Time VELOCITY 3 µmol / min or Rate 1 min
15 µmol S vs 1 µmol E
Time
[Product]
Slope = Initial velocity
= v0 = [P] / time
2 min
3 µmol / min
4 min
<3 µmol / min
CHMI E.R. Gauthier, Ph.D.

7. Enzymatic reactions v0 is proportional to [E] 3µmol E 2µmol E
1 min
3 µmol / min
15 µmol S vs 1 µmol E
6 µmol / min
15 µmol S vs 2 µmol E
9 µmol / min
15 µmol S vs 3 µmol E
Time
[Product]
v0 is proportional to [E]
1µmol E
2µmol E
3µmol E
CHMI E.R. Gauthier, Ph.D.

8. Enzymatic reactions Maximum velocity = Vmax Vmax ½ Vmax v0 [Substrate]
1 µmol / min
1 min
[Substrate] v0
Maximum velocity = Vmax
Vmax
½ Vmax
2 µmol / min
1 min
3 µmol / min
1 min
E saturated by S
CHMI E.R. Gauthier, Ph.D.

9. Enzymatic reactions
CHMI E.R. Gauthier, Ph.D.

10. Michaelis-Menten Equation
[Substrate] v0
Maximum velocity = Vmax
Vmax
½ Vmax
vo = Vmax [S]
Km + [S]
CHMI E.R. Gauthier, Ph.D.

11. Michaelis-Menten Equation
[Substrate] v0
Vmax
½ Vmax
Km1
Km2 E2 E1
CHMI E.R. Gauthier, Ph.D.

12. Km
CHMI E.R. Gauthier, Ph.D.

13. Turnover number E + S ES E + P FAST SLOW k1 k2 K-1
CHMI E.R. Gauthier, Ph.D.

14. Measuring Km and Vmax Vmax ½ Vmax v0 Km [Substrate]
CHMI E.R. Gauthier, Ph.D.

15. Measuring Km and Vmax 1/vo 1 = Km x 1 + 1 vo Vmax [S] 1/Vmax 1/[S]
Lineweaver-Burk plot 1 vo
= Km x
Vmax
[S]
CHMI E.R. Gauthier, Ph.D.