Download Enz03 kinetic parameters

Enzyme Kinetics
kcat /Km
Vmax [S]
vo=
Km + [S]
kcat
Significance
zero order
1st order
Observe vo change
under various [S],
resulted plots
yield Vmax and Km
Turn over
number
Vmax
k3 [Et]
1 mmole
min
Specific Activity
Activity
&
E3
E2
E1
Km
Affinity with
substrate
Double reciprocal
Bi-substrate reaction also
follows M-M equation, but
one of the substrate should
be saturated when estimate
the other
Inhibition
Maximum
velocity
Activity Unit
unit
mg
Direct plot
Competitive
Non-competitive
Uncompetitive
Juang RH (2004) BCbasics
Significance of Enzyme Kinetics
Obtain
Vmax and Km
1st order
[S] = Low → High
vo =
= k3 [Et] × K
Vmax [S]
Km + [S]
E3
E2
E1
Proportional to
enzyme concentration
zero order
v0 = Vmax × K
[S] = Fixed concentration
Juang RH (2004) BCbasics
Km: Affinity with Substrate
Vmax [S]
vo =
Km + [S]
When using different substrate
2
S2
S1
Vmax
=
Vmax [S]
Km + [S]
S3
1/2
Km + [S] = 2 [S]
S1 S2 S3
Km
Affinity changes
Km = [S]
Juang RH (2004) BCbasics
Vmax
Vmax
If vo =
2
Km: Hexokinase Example
Glucose + ATP → Glc-6-P + ADP
Glucose
number
CHO
1
H-C-OH
2
HO-C-H
3
H-C-OH
4
H-C-OH
5
6
H2-C-OH
Km =
8
Allose
CHO
H-C-OH
H-C-OH
H-C-OH
H-C-OH
H2-C-OH
8,000
Mannose Substrate
CHO
HO-C-H
HO-C-H
H-C-OH
H-C-OH
H2-C-OH
5 mM
Juang RH (2004) BCbasics
Turn Over Number, kcat
E+S
k1
k2
k3
ES (v ) E + P
o
When substrate excess, k3 = kcat, turn over number (t.o.n)
When [substrate] is low
vo =
Vmax [S]
Km + [S]
Start from M-M eq.
Second order
(IV)
=
k3 [E][S]
Km + [S]
=
Omit the [substrate]
k3
Km
[E][S]
Substrate specificity
Juang RH (2004) BCbasics
Turn Over Numbers of Enzymes
kcat (s-1)
Enzymes
Substrate
Catalase
H2O2
Carbonic anhydrase
HCO3-
400,000
Acetylcholinesterase
Acetylcholine
140,000
b-Lactamase
Benzylpenicillin
Fumarase
Fumarate
RecA protein (ATPase)
ATP
40,000,000
2,000
800
0.4
The number of product transformed from substrate
by one enzyme molecule in one second
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.263
Chymotrypsin Has Distinct kcat /Km to Different Substrates
=
–
– –
=
O
R O
H3C–C–N–C–C–O–CH3
H H
kcat / Km
R=
Glycine
–H
1.3 ╳ 10-1
Norvaline
–CH2–CH2–CH3
3.6 ╳ 102
Norleucine
–CH2–CH2–CH2–CH3
3.0 ╳ 103
Phenylalanine –CH2–
1.0 ╳ 105
(M-1 s-1)
Adapted from Mathews et al (2000) Biochemistry (3e) p.379
t
vo = [P] / min
Unit = mmole/min
Slope
tan
0
10
20 30
40
Reaction time (min)
Specific Activity Units
Activity = Protein (mg)
y
x
y
= tan
x
Juang RH (2004) BCbasics
S → P mmole
Product [P]
Enzyme Activity Unit