Inhibition of Enzyme Activity
Types of Inhibition: Competitive Noncompetitive Uncompetitive Product Inhibition Suicide Inhibition
Competitive Inhibition
Fig 8-15
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 +I c
-I c
1 / Vo
+Ic
Vo
y-int = 1/Vmax
-I c
So x-int = -1/Km
1 / So slope = K m /Vmax
c
yint = -1 / (Km . (1+ [I
] / K c ))
Noncompetitive Inhibition
Fig 8-15
Noncompetitive Inhibition
NONCOMPETITIVE -Inc -Inc Equilibria Scheme P + E 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
(inactive)
- I nc + I nc
slope = Km . (1+ [Inc ] / K c ) / Vmax +I 1 / Vo -I
Vo
So y-int = (1+[I nc] / Knc ) / Vmax x-int = -1/Km 1 / So slope = K m /Vmax
y-int = 1/Vmax
Examples: Competitive and Noncompetitive Inhibition
H+ H2N O H H C N pyruvate R **NADH (reduced form) R **NAD
(oxidized form)
O + CH3 C CO2-
O C NH2
OH + CH3 C CO2H L-lactate
ORDERED BI BI MECHANISM
NADH PYR LAC NAD
+
S1
S2
P1
P2
ES1
ES1S2
EP1P2
EP2
Examples: Competitive and Noncompetitive Inhibition
LACTATE DEHYDROGENASE O H H H2N C N pyruvate R **NADH (reduced form) HO H O C NH 2 N R NAD-OH H
+
O + CH3 C CO2-
O C NH2 + CH3
N R **NAD
(oxidized form)
OH C CO2H
L-lactate
O H2N C CO2oxamate
INHIBITORS:
O CH3CH2HN C CO2ethyloxamate
For multisubstrate rxns, the type of inhibition depends upon the substrate that is varied in the inhibition experiment!
LACTATE DEHYDROGENASE O H H H2N C N pyruvate R **NADH (reduced form) HO H O C NH 2 N R NAD-OH H+ H CO2N R **NAD
(oxidized form)
O + CH3 C
O C NH2
OH + CH3 C CO2H L-lactate
INHIBITORS:
What substrate does this inhibitor resemble?
NADH
Which plot describes the inhibition of lactate dehydrogenase by NAD-OH when NADH is the varied substrate?
O H H HNC
2
H+
O CH3 C CO2pyruvate HO H O C N R NAD-OH
O C NH2
ORDERED BI BI MECHANISM
N R **NADH
(reduced form)
N R **NAD
(oxidized form)
OH + CH3 C CO2H L-lactate
NADH
PYR
LAC
NAD+
S1
S2
P1
P2
ES1
ES1S2
EP1P2
EP2
NH2
Competitive inhibition seen if varied S is NADH
Competitive +I 1 / Vo -I -I
Noncompetitive +I 1 / Vo
1/NADH
1/NADH
Which plot describes the inhibition of lactate dehydrogenase by NAD-OH when pyruvate is the varied substrate?
O H H HNC
2
H+
O CH3 C CO2pyruvate HO H O C N R NAD-OH
O C NH2
ORDERED BI BI MECHANISM
N R **NADH
(reduced form)
N R **NAD
(oxidized form)
OH + CH3 C CO2H L-lactate
NADH
PYR
LAC
NAD+
S1
S2
P1
P2
ES1
ES1S2
EP1P2
EP2
NH2
Noncompetitive inhibition seen if varied S is pyruvate
Competitive
1 / Vo -I +I +I
Noncompetitive
1 / Vo
-I
1/PYRUVATE
1/PYRUVATE
Which plot describes the inhibition of lactate dehydrogenase by oxamate when NADH is the varied substrate?
O H H HNC
2
H+
O CH3 C CO2pyruvate
O C NH2
ORDERED BI BI MECHANISM
N R **NADH
(reduced form)
OH + CH3 C CO2H L-lactate
NADH
PYR
LAC
NAD+
S1
S2
P1
P2
O H2N C CO2oxamate
R **NAD
(oxidized form)
ES1
ES1S2
EP1P2
EP2
Noncompetitive inhibition seen if varied S is NADH
Competitive +I 1 / Vo -I -I
Noncompetitive +I 1 / Vo
1/NADH
1/NADH
Which plot describes the inhibition of lactate dehydrogenase by oxamate when pyruvate is the varied substrate?
O H H HNC
2
H+
O CH3 C CO2pyruvate
O C NH2
ORDERED BI BI MECHANISM
N R **NADH
(reduced form)
OH + CH3 C CO2H L-lactate
NADH
PYR
LAC
NAD+
S1
S2
P1
P2
O H2N C CO2oxamate
R **NAD
(oxidized form)
ES1
ES1S2
EP1P2
EP2
Noncompetitive
+I 1 / Vo -I
Competitive inhibition seen if varied S is pyruvate
Competitive
1 / Vo +I
-I
1/PYRUVATE
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
UNCOMPETITIVE -Iu -Iu -Iu Equilibria Scheme P+ E 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
y-int = (1+ [Iu] / Ku ) / V max
+ Iu
1/Vo Vo
- Iu
+I -I slope = Km / Vmax
So x-int = -(1+ [Iu] / Ku ) / Km x-int = -1 / Km
1 / So y-int = 1 / Vmax
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 C N R **NAD
(oxid ized fo rm )
O NH2 O C H + H C OH + HO P OOH CH2OPi
O H H H2N C N R **NADH
(red uce d fo rm)
O +
C O P-O O H C OH CH2OPi
g lyceraldeh yd e-3-Pi
1,3-b isphospho glycer ate
O INHIBITOR: HO As OOH
ORDERED TRI BI MECHANISM
NAD + GAP Pi 1,3-BPG N ADH
S1 S2 S3
P1
P2
ES 1
ES' 1 P 2
ES' 1 P 2
EP 1 P 2
EP 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 C N R **NAD
(oxid ized fo rm )
O NH2 O C H + H C OH + HO P OOH CH2OPi
O H H H2N C N R **NADH
(red uce d fo rm)
C O P-O O H C OH CH2OPi
g lyceraldeh yd e-3-Pi
1,3-b isphospho glycer ate
O INHIBITOR: HO As OOH
ORDERED TRI BI MECHANISM
NAD + GAP Pi 1,3-BPG N ADH
S1 S2 S3
P1
P2
ES 1
ES' 1 P 2
ES' 1 P 2
EP 1 P 2
EP 2
Predict the type of inhibition by H2 AsO4 - when each of the substrates is varied in inhibition experiments
ORDERED TRI BI MECHANISM
NAD + GAP Pi
O
NADH
O HO P OOH
1,3-BPG
S1 S 2 S 3
P1
P2
HO As OOH
ES1
ES'1P2
ES' 1P2
EP1P2
EP2
.
Noncompetitive +I
1 / Vo -I 1 / Vo -I
Competitive +I
1 / Vo
Uncompetitive +I
-I
1/So
1/S o
1/S o
If So = Pi
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 Vo slope = Km / Vmax So x-int = -1 / Km 1 / So P+ E
Example: Product Inhibition
-galactosidase (lactase)
lactose HOCH2 HO O OH O OH HOCH2 HO O OH OH galactose OH HO OH glucose H H HOCH2 O OH OH O OH HOCH2 O OH OH
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 Vo -I +I -I
So
1 / So
Michaelis-Menten and Lineweaver-Burk plots look noncompetitive
10
Suicide Inhibition
+I 1 / Vo Vo -I +I -I
So
1 / So
k1 E + S k -1 Nu: + R-X ( = I) X E Nu R inactived enzyme ES
k2
P + E
The suicide inhibitor removes E so that the [ES] is lower, Vmax is lower, and inhibition cannot be overcome at high So
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 k -1 Nu: + R-X ( = I) X E Nu R inactived enzyme -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 ES k2 P + E
O CHY-HIS=N :
O CH3 Cl (X) CHY-HIS=N-
Cl CH2 C CH NH S CH2 O
irreversibly inactivated
Suicide Inhibitors: Aspirin
Drug
CO2H O C CH 3
Target Enzymes
(Box 21-1)
Aspirin
CO2H CH CH3
aspirin
CO2Na
Cyclooxygenases 1 and 2
O C CH 3 NH
CH CH3
"Simple" Reversible Inhibitors
CH2 CH CH3 CH3
ibuprofen
O CH3
naproxen
OH
tylenol O CH3 CNHCHCO2 CH2 N-acetylcysteine antidote for SH tylenol poisoning
[Link]
12
Cyclooxygenase and Modification by Aspirin
[Link] .html
CO2H
+ Cox-1 O C CH 3 CH2OH
aspirin
CO2H
+
OH
salicylic acid
Cox-1 CH2O
C CH 3 O
[Link]
Suicide Inhibitors: New NSAIDs
Drug Celebrex Vioxx Target Enzyme Cyclooxygenase 2
Tylenol and Vioxx, two other medications commonly used for arthritis, were similarly tested Both groups showed no competitive interaction with aspirin. [Link]
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
[Link]/chem-dept/office/jwolfe/[Link]
Cl
Cl O
Clorgyline N
Deprenyl N
Pargyline N
[Link]
14