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5 Enzyme
5 Enzyme
Types of Inhibition:
Competitive
Noncompetitive
Uncompetitive
Product Inhibition
Suicide Inhibition
Competitive Inhibition
Fig 8-15
Competitive Inhibition
COMPETITIVE
Equilibria Scheme
+Ic
Vo
+I c
1 / Vo
-I c
-I c
y-int = 1/Vmax
So
1 / So
x-int = -1/Km
slope = K m /Vmax
] / K c ))
Noncompetitive Inhibition
Fig 8-15
Noncompetitive Inhibition
NONCOMPETITIVE
Equilibria Scheme
E + S
ES
is not structurally similar to S; is not an S
Km
+
binds to free E or ES at a site where S
+
I nc
I nc
does not bind
-Inc does NOT compete with S for free E
K nc
K'nc
-High S cannot overcome inhibition because Inc
binds to ES complex, inactivating it
EI nc + S
ESI nc
P + E
-Inc
-Inc
(inactive)
- I nc
+ I nc
1 / Vo
Vo
-I
So
1 / So
slope = K m /Vmax
x-int = -1/Km
y-int = 1/Vmax
O H H
C
CO2-
CH3 C
OH
+ CH3 C CO2H
NH2
L-lactate
pyruvate
R
**NADH
(reduced form)
R
**NAD
(oxidized form)
ORDERED BI BI MECHANISM
+
NADH
PYR
LAC
NAD
S1
S2
P1
P2
ES1
ES1S2
EP1P2
EP2
O H H
H2N C
O
+
CH3 C
CO2-
INHIBITORS:
NH2
pyruvate
R
**NADH
(reduced form)
O
+ CH3
OH
C CO2H
L-lactate
R
**NAD
(oxidized form)
HO H O
C NH
2
N
O
H2N C CO2-
O
CH3CH2HN C CO2-
oxamate
ethyloxamate
R
NAD-OH
O H H
H2N C
O
+
CH3 C
pyruvate
R
**NADH
(reduced form)
INHIBITORS:
CO2-
O
NH2
OH
+ CH3 C CO2H
L-lactate
R
**NAD
(oxidized form)
HO H O
C NH
2
N
R
NAD-OH
NADH
O H H
HNC
O
CH3 C CO2-
ORDERED BI BI MECHANISM
O
NH2
N
HO H O
C
(reduced form)
NADH
PYR
LAC
NAD+
S1
S2
P1
P2
L-lactate
pyruvate
R
**NADH
OH
+ CH3 C CO2H
R
E
**NAD
ES1
ES1S2
(oxidized form)
EP2
EP1P2
NH2
Competitive inhibition
seen if varied S is NADH
R
NAD-OH
Competitive
+I
Noncompetitive
+I
1 / Vo
1 / Vo
-I
-I
1/NADH
1/NADH
O H H
HNC
O
CH3 C CO2-
ORDERED BI BI MECHANISM
O
C
NH2
(reduced form)
HO H O
C
N
R
NAD-OH
NADH
PYR
LAC
NAD+
S1
S2
P1
P2
L-lactate
pyruvate
R
**NADH
OH
+ CH3 C CO2H
R
E
**NAD
(oxidized form)
ES1
ES1S2
EP2
EP1P2
NH2
Noncompetitive inhibition
seen if varied S is pyruvate
Noncompetitive
Competitive
+I
1 / Vo
1 / Vo
-I
1/PYRUVATE
+I
-I
1/PYRUVATE
O H H
HNC
O
CH3 C CO2-
ORDERED BI BI MECHANISM
O
NH2
PYR
LAC
NAD+
S1
S2
P1
P2
(reduced form)
NADH
L-lactate
pyruvate
R
**NADH
OH
+ CH3 C CO2H
H2N C CO2oxamate
ES1
**NAD
ES1S2
(oxidized form)
EP2
EP1P2
Noncompetitive inhibition
seen if varied S is NADH
Competitive
+I
Noncompetitive
+I
1 / Vo
1 / Vo
-I
-I
1/NADH
1/NADH
O H H
HNC
O
CH3 C CO2-
ORDERED BI BI MECHANISM
O
C
NH2
(reduced form)
H2N C CO2-
NADH
PYR
LAC
NAD+
S1
S2
P1
P2
L-lactate
pyruvate
R
**NADH
OH
+ CH3 C CO2H
R
E
**NAD
(oxidized form)
ES1
ES1S2
EP2
EP1P2
oxamate
Noncompetitive
Competitive
+I
1 / Vo
1 / Vo
-I
1/PYRUVATE
+I
-I
1/PYRUVATE
Uncompetitive Inhibition
This type of inhibition requires that one or more
substrates bind to E before the inhibitor can bind
Uncompetitive Inhibition
This type of inhibition requires that one or more
substrates bind to E before the inhibitor can bind
Equilibria Scheme
UNCOMPETITIVE
-Iu
-Iu
-Iu
P+ E
1/Vo
+I
-I
- Iu
Vo
slope = Km / Vmax
1 / So
So
x-int = -(1+ [Iu] / Ku ) / Km
x-int = -1 / Km
y-int = 1 / Vmax
O
C
NH2
N
R
**NAD
O H H
H2N C
O
C H
+ H C OH + HO P OOH
CH2OPi
R
**NADH
(oxid ized fo rm )
HO As OOH
C O P-O
O
H C OH
CH2OPi
O
INHIBITOR:
g lyceraldeh yd e-3-Pi
GAP
Pi
ES 1
N ADH
1,3-BPG
P1
S1 S2 S3
ES' 1 P 2
ES' 1 P 2
P2
EP 2
EP 1 P 2
O
C
NH2
N
R
**NAD
O H H
H2N C
O
C H
+ H C OH + HO P OOH
CH2OPi
R
**NADH
(oxid ized fo rm )
HO As OOH
C O P-O
O
H C OH
CH2OPi
O
INHIBITOR:
g lyceraldeh yd e-3-Pi
GAP
Pi
1,3-BPG
P1
S1 S2 S3
ES 1
ES' 1 P 2
ES' 1 P 2
EP 1 P 2
N ADH
P2
EP 2
GAP
Pi
S1 S 2 S 3
O
NADH
1,3-BPG
P1
HO P O-
HO As O-
P2
OH
OH
ES'1P2
ES1
ES' 1P2
EP1P2
EP2
.
Competitive
+I
Noncompetitive
+I
Uncompetitive
1 / Vo
1 / Vo
1 / Vo
-I
+I
-I
-I
1/S o
1/So
If So = Pi
1/S o
If So = NADH or GAP
Product Inhibition
Equilibria Scheme
PRODUCT INHIBITION
E + S
ES
Km
-Ip is structurally similar to S
+
-Ip binds to free E at active site where S binds
P
-Ip competes with S for free E
Kp
-At low S, resembles competitive inhibition
-However, at high S, the inhibition is not overcome
EIp
because higher levels of P are generated which
inhibit the enzyme
1 / Vo
P+ E
Vo
slope = Km / Vmax
So
1 / So
x-int = -1 / Km
lactose
O
OH
HOCH2
O
HO
OH
OH
OH
O
OH
HOCH2
HOCH2
O
OH
HO
OH
O
OH
OH
HO
OH
OH
glucose
galactose
Suicide Inhibition
This type of enzyme inhibition results in the stoichiometric covalent modification of a
side chain on an amino acid in the active site of an enzyme. The inhibitor chemically
resembles a (one of the) substrate(s) and binds in the active site in the same way as the
substrate(s) binds. The inhibitor, however, has a functional group, ususally a leaving group,
that is replaced by a nucleophile in the enzyme active site. This covalent enzyme-inhibitor
complex forms irreversibly, thereby irreversibly inactivating the enzyme. Therefore this
type of inhibition is called "suicide inhibition" or affinity labeling and the inhibitor is called a
"suicide inhibitor". This reaction with the suicide inhibitor removes active enzyme from
the system; this removal is measured as inhibition. Since active enzyme is lost, the inhibition
is not relieved at high substrate levels. The rate, at high substrate in the presence of the
inhibitor,is still proportional to the amount of the enzyme-substrate complex. However, the
maximum amount of that complex is limited by the remaining amount of active enzyme, not by
the total enzyme added to the system.
+I
1 / Vo
-I
+I
Vo
So
-I
1 / So
10
Suicide Inhibition
+I
1 / Vo
-I
+I
Vo
-I
So
1 / So
k1
E + S
ES
k2
P + E
k -1
Nu:
+
R-X ( = I)
X
E
Nu
R
inactived enzyme
+I
1 / Vo
-I
Chymotrypsin Inhibitor
tosyl phenylalanyl
chloromethylketone
1 / So
tpck
11
E + S
ES
k2
P + E
k -1
Nu:
+
R-X ( = I)
X
E
Nu
R
inactived enzyme
Cl CH2 C CH NH S
CHY-HIS=N :
CH3
CHY-HIS=N-
CH2
Cl
(X)
irreversibly
inactivated
Drug
O C CH 3
Aspirin
aspirin
CO2H
CO2Na
CH CH3
CH CH3
Target Enzymes
(Box 21-1)
Cyclooxygenases 1 and 2
O
C CH 3
NH
"Simple"
Reversible
Inhibitors
CH2
CH
CH3
CH3
ibuprofen
O
CH3
naproxen
http://cti.itc.virginia.edu/~cmg/Demo/pdb/cycox/cycox_2.html
OH
tylenol
O
CH3 CNHCHCO2 CH2
N-acetylcysteine
antidote for
SH
tylenol poisoning
12
CO2H
+ Cox-1
O C CH 3
CH2OH
aspirin
CO2H
Cox-1
OH
salicylic acid
CH2O
C CH 3
O
http://www.scripps.edu/pub/goodsell/pdb/pdb17/pdb17_1.html
Target Enzyme
Vioxx
Cyclooxygenase 2
13
Cl
Cl
Clorgyline
Deprenyl
N
Pargyline
N
http://www.csusm.edu/DandB/AD.html
14