lecture 15 tuesday 3/4/08 enzymes michealis-menten kinetics lineweaver-burk plot enzyme inhibition
Post on 22-Dec-2015
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Vmax
=kcat
* Et
• Turnover Number: kcat
Number of substrate molecules (moles) converted to product
in a given time (s) on a single enzyme molecule
(molecules/molecule/time)
H2O2 + E H2O + O + E
40,000,000 molecules of H2O2 converted to product per second on a single enzyme
molecule.
Enzymes, InhibitionS + E → E + P
1) Mechanism and active intermediate (E·S)
2) Rates
3) Net Rates
EPOHSE
SESE
SESE
k
k
k
3
2
1
2 3
2
1
SOHSEP
OHSESE
SESE
ESSE
rCCkr
CCkr
Ckr
CCkr
2
2
3
33
22
11
SESESESE rrrr 321
4) Pseudo Steady-State
SOH
SEtotOH
OH
SOH
EtotSOHP
OH
S
EtotEE
OH
ESESEEtot
OH
ESOHP
OH
ES
OHSESEES
Ck
Ckk
CCCk
CkkCk
Ckk
CCCkkr
CkkCk
CCC
Ckk
CCkCCC
Ckk
CCCkkr
Ckk
CCkSE
CCkCkCCk
1
32
3
32
132
31
32
132
1
32
31
32
1
321
2
2
2
2
2
2
2
2
2
2
2
1
1
0
(Michaelis-Menten plot)
Vmax
-rs
S1/2
2/1
2/1maxmaxmax
1
32
max3
2
constant) (michaelis
2
2
Sk
SVVr
Ck
CVr
k
Ckkk
VCkCkk
mS
Sm
SS
OHm
EtotcatOHcat
km=S1/2 therefore km is the concentration at which the rate is half the maximum rate
Competitive Inhibition
E + S E·S E + P
+ I E · I
1) Mechanisms
2) Rates
k1
k2
k3
k4
k5
SEP Ckr
IEIE
IEIEEPSE
SESESESE
3
m
ESESSE
SESEESSE
k
CC
kk
CCkC
CkCkCCkr
32
1
3210
4
5EI
54EI3
0r
k
kk
k
CCC
CkCCkk
CCkr
II
EI
EIEIm
ESP
SI
Im
S
I
ImS
SS
I
mISm
SEtotP
I
I
m
S
EtotEEISEEEtot
Ck
C
V
k
Vr
k
CkC
CVr
k
kCCk
CCkr
k
C
k
CC
CCCCC
11
11
1
1
maxmax
max3
From before (no competition):S
m
S CV
k
Vr
111
maxmax
Intercept does not change, slope increases as inhibitor concentration increases
max
slopeV
km
max
1Intercept
V
Sr
1
SC
1
No competition
Competition