Inhibition of Enzyme Activity
Types of Inhibition:
Competitive
Noncompetitive
Uncompetitive
Product Inhibition
Suicide Inhibition
Competitive Inhibition
Fig 8-15
1
Competitive Inhibition
COMPETITIVE
Equilibria Scheme
-Ic structrually resembles S, but is not an S
E + S
ES
P + E
Km
-Ic binds to free E at active site where S binds
+
-Ic competes with S for free E
Ic
-High S overcomes inhibition because all E
is bound in ES complex; since rate [ES] and Kc
[ES] is max, rate is max; no EI c is present
EI c slope = K m . (1+ [I c ] / K c ) / Vma x
+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
yint = -1 / (Km . (1+ [I
c
] / K c ))
Noncompetitive Inhibition
Fig 8-15
2
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
slope = Km . (1+ [Inc ] / Kc ) / Vmax
+I
+ I nc
1 / Vo
Vo
-I
So
1 / So
y-int = (1+[I nc] / Knc ) / Vmax
slope = K m /Vmax
x-int = -1/Km
y-int = 1/Vmax
Examples: Competitive and Noncompetitive Inhibition
H+
H2N
O H H
C
+
O
H
O
C
CO2-
CH3 C
N
OH
+ CH3 C CO2H
NH2
N
L-lactate
pyruvate
R
**NADH
(reduced form)
R
**NAD
(oxidized form)
ORDERED BI BI MECHANISM
+
NADH
PYR
LAC
NAD
S1
S2
P1
P2
E
ES1
ES1S2
EP1P2
EP2
E
3
Examples: Competitive and Noncompetitive Inhibition
LACTATE DEHYDROGENASE
+
H
O H H
H2N C
H
O
+
CH3 C
C
CO2-
N
INHIBITORS:
NH2
N
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
For multisubstrate rxns, the type of inhibition depends upon
the substrate that is varied in the inhibition experiment!
LACTATE DEHYDROGENASE
H+
O H H
H2N C
H
O
+
CH3 C
pyruvate
R
**NADH
(reduced form)
INHIBITORS:
C
CO2-
N
O
NH2
N
OH
+ CH3 C CO2H
L-lactate
R
**NAD
(oxidized form)
HO H O
C NH
2
What substrate does this
inhibitor resemble?
N
R
NAD-OH
NADH
4
Which plot describes the inhibition of lactate dehydrogenase
by NAD-OH when NADH is the varied substrate?
H+
O H H
HNC
+
2
H
O
CH3 C CO2-
C
N
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
E
NH2
N
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
Which plot describes the inhibition of lactate dehydrogenase
by NAD-OH when pyruvate is the varied substrate?
H+
O H H
HNC
+
2
O
CH3 C CO2-
N
H
ORDERED BI BI MECHANISM
O
C
NH2
N
(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
E
NH2
Noncompetitive inhibition
seen if varied S is pyruvate
Noncompetitive
Competitive
+I
1 / Vo
1 / Vo
-I
1/PYRUVATE
+I
-I
1/PYRUVATE
5
Which plot describes the inhibition of lactate dehydrogenase
by oxamate when NADH is the varied substrate?
H+
O H H
HNC
+
2
H
O
CH3 C CO2-
C
N
ORDERED BI BI MECHANISM
O
NH2
N
PYR
LAC
NAD+
S1
S2
P1
P2
R
O
(reduced form)
NADH
L-lactate
pyruvate
R
**NADH
OH
+ CH3 C CO2H
H2N C CO2oxamate
ES1
E
**NAD
ES1S2
(oxidized form)
EP2
EP1P2
E
Noncompetitive inhibition
seen if varied S is NADH
Competitive
+I
Noncompetitive
+I
1 / Vo
1 / Vo
-I
-I
1/NADH
1/NADH
Which plot describes the inhibition of lactate dehydrogenase
by oxamate when pyruvate is the varied substrate?
H+
O H H
HNC
+
2
O
CH3 C CO2-
N
H
ORDERED BI BI MECHANISM
O
C
NH2
N
O
(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
E
oxamate
Competitive inhibition seen
if varied S is pyruvate
Noncompetitive
Competitive
+I
1 / Vo
1 / Vo
-I
1/PYRUVATE
+I
-I
1/PYRUVATE
6
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
is not structurally similar to S; is not an S
E + S
ES
Km
binds to ES only; S opens up a site for
u I
+
binding site may be in active site but binding
Iu
of Iu requires prior binding of S
Ku
-High S cannot overcome inhibition because presence
uI
of S is required to provide a site for binding of
EIu
P+ E
y-int = (1+ [Iu] / Ku ) / V max
+ Iu
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
7
Example: Uncompetitive Inhibition
This type of inhibition requires that one or more
substrates bind to E before the inhibitor can bind
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE
H
O
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
1,3-b isphospho glycer ate
(red uce d fo rm)
O
INHIBITOR:
+
N
g lyceraldeh yd e-3-Pi
O
O
ORDERED TRI BI MECHANISM
NAD +
GAP
Pi
E
ES 1
N ADH
1,3-BPG
P1
S1 S2 S3
ES' 1 P 2
ES' 1 P 2
P2
E
EP 2
EP 1 P 2
.
Predict the type of inhibition by H2 AsO4 - when each of the
substrates is varied in inhibition experiments
This type of inhibition requires that one or more
substrates bind to E before the inhibitor can bind
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE
H
O
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
1,3-b isphospho glycer ate
(red uce d fo rm)
O
INHIBITOR:
+
N
g lyceraldeh yd e-3-Pi
O
O
ORDERED TRI BI MECHANISM
NAD +
GAP
Pi
1,3-BPG
P1
S1 S2 S3
E
ES 1
ES' 1 P 2
ES' 1 P 2
EP 1 P 2
N ADH
P2
EP 2
E
.
8
Predict the type of inhibition by H2 AsO4 - when each of
the substrates is varied in inhibition experiments
ORDERED TRI BI MECHANISM
NAD +
GAP
O
Pi
S1 S 2 S 3
O
NADH
1,3-BPG
P1
HO P O-
HO As O-
P2
OH
OH
E
ES'1P2
ES1
ES' 1P2
EP1P2
EP2
E
.
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
9
Example: Product Inhibition
β -galactosidase (lactase)
HOCH2
lactose
O
OH
HOCH2
O
HO
OH
O
OH
OH
O
OH
H
H
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
Michaelis-Menten and Lineweaver-Burk plots “look” noncompetitive
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
The suicide inhibitor removes E so that the
[ES] is lower, Vmax is lower, and inhibition
cannot be overcome at high So
Nu
R
inactived enzyme
Example: Suicide Inhibition with Chymotrypsin
One synthetic substrate for chymotrypsin
+I
1 / Vo
-I
Chymotrypsin Inhibitor
tosyl phenylalanyl
chloromethylketone
1 / So
tpck
11
Example: Suicide Inhibition with Chymotrypsin
k1
E + S
ES
k2
P + E
k -1
Nu:
+
R-X ( = I)
-at all So, Vo % [ES]
-I chemically resembles S (I added first, S second to start reaction)
-I reacts 1:1 with enzyme usually
-I lowers [E], therefore lowering [ES] and Vo
-I lowers maximum amount of [ES] because it removes E
-V o can never reach Vmax
-resembles noncompetitive inhibition because inhibition cannot
be relieved at high S
X
E
Nu
R
inactived enzyme
O
O
Cl CH2 C CH NH S
CHY-HIS=N :
CH3
CHY-HIS=N-
O
CH2
Cl
(X)
R
irreversibly
inactivated
Suicide Inhibitors: Aspirin
CO2H
Drug
O C CH 3
Aspirin
aspirin
O
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
Cyclooxygenase and Modification by Aspirin
http://www.chem.uwec.edu/Webpapers_F98/bruce/Pages/aspcommands .html
CO2H
+ Cox-1
O C CH 3
CH2OH
aspirin
O
CO2H
+
Cox-1
OH
salicylic acid
CH2O
C CH 3
O
http://www.scripps.edu/pub/goodsell/pdb/pdb17/pdb17_1.html
Suicide Inhibitors: New NSAIDs
Drug
Celebrex
Target Enzyme
Vioxx
Cyclooxygenase 2
Tylenol and Vioxx, two other medications
commonly used for arthritis, were similarly tested
… Both groups showed no competitive
interaction with aspirin.
http://www.pslgroup.com/dg/1e8fa2.htm
Two phases of inhibition:
1. Rapid competitive inhibition
2. Slower irreversible inhibition (covalent modification)
13
Suicide Inhibitors: Inhibitors of monoamine
oxidase (MAO) for treatment of depression
www.chem.vt.edu/chem-dept/office/jwolfe/DRUGCHEM1.ppt
Cl
Cl
Clorgyline
O
N
Deprenyl
N
Pargyline
N
http://www.csusm.edu/DandB/AD.html
14