CHMI 2227E
Biochemistry I
Enzymes:
-
Kinetics
CHMI 2227 - E.R. Gauthier, Ph.D.
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Enzymatic reactions
Enzyme (each = 1 µmol)
Substrate (each = 1 µmol)
X min
Only concentrations we know  we’re
the ones who set up the experiment!
Product
CHMI 2227 - E.R. Gauthier, Ph.D.
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Enzymatic reactions
How do we measure enzyme activity?

1. Detection of the product(s):

pNA = para-nitroaniline  Absorbs at 405 nm
O
DEVD-pNA
(uncolored)
O
O
O
H3+N-CH-C-NH-CH-C-NH-CH-C-NH-CH-C-NHCH2
COO-
CH2
CH
CH2 H3C CH3
COO-
CH2
COOMeasure increase
in A405nm
Caspase 3 (proteasehydrolase)
O
O
NO2
O
O
DEVD
H3+N-CH-C-NH-CH-C-NH-CH-C-NH-CH-C-OH
(uncoloured)
CH2
CH2
CH
CH2
COOCH2 H3C CH3
COOCOO
CHMI 2227 - E.R. Gauthier, Ph.D.
H2N
NO2
pNA (yellow)
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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
Lactate
dehydrogenase
CHMI 2227 - E.R. Gauthier, Ph.D.
Measure decrease
in A340nm
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Enzymatic reactions
How do we measure enzyme activity?

3. Coupled reactions:

Very useful when neither substrate/product/co-factor can be
(easily) detected;
Detectable by HPLC
but not practical
Glutaminase
1st reaction
+ NH4+
2nd reaction
+ NAD+
+ H2O
Glutamate
Dehydrogenase
+ NADH +H+
+ NH4+
CHMI 2227 - E.R. Gauthier, Ph.D.
Measure increase
in A340nm
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Enzymatic reactions
3 µmol / min
VELOCITY
or Rate
1 min
Slope = Initial velocity
= v0 = [P] / time
3 µmol / min
2 min
<3 µmol / min
[Product]
15 µmol S vs 1 µmol E
Time
4 min
CHMI 2227 - E.R. Gauthier, Ph.D.
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Enzymatic reactions
3 µmol / min
1 min
v0 is proportional to [E]
15 µmol S vs 1 µmol E
6 µmol / min
1 min
[Product]
3µmol E
2µmol E
1µmol E
15 µmol S vs 2 µmol E
9 µmol / min
Time
1 min
15 µmol S vs 3 µmol E
CHMI 2227 - E.R. Gauthier, Ph.D.
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Enzymatic reactions
1 µmol / min
1 min
Maximum velocity = Vmax
Vmax
2 µmol / min
1 min
v0
½ Vmax
3 µmol / min
[Substrate]
1 min
E saturated by S
CHMI 2227 - E.R. Gauthier, Ph.D.
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Enzymatic reactions
CHMI 2227 - E.R. Gauthier, Ph.D.
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Michaelis-Menten Equation
Maximum velocity = Vmax
Vmax
v0
½ Vmax
[Substrate]
vo = Vmax [S]
Km + [S]
CHMI 2227 - E.R. Gauthier, Ph.D.
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Michaelis-Menten Equation
E1
Vmax
E2
v0
½ Vmax
Km1 Km2
[Substrate]
CHMI 2227 - E.R. Gauthier, Ph.D.
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Km
CHMI 2227 - E.R. Gauthier, Ph.D.
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Turnover number
E +S
k1
K-1
FAST
CHMI 2227 - E.R. Gauthier, Ph.D.
ES
k2
E+P
SLOW
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Measuring Km and Vmax
Vmax
v0
½ Vmax
Km
CHMI 2227 - E.R. Gauthier, Ph.D.
[Substrate]
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Measuring Km and Vmax
1/vo
1 = Km
vo
Vmax
x 1
[S]
+
1
Vmax
1/Vmax
1/[S]
-1/Km
Lineweaver-Burk plot
CHMI 2227 - E.R. Gauthier, Ph.D.
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