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

+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

] / 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

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

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

Examples: Competitive and Noncompetitive Inhibition

LACTATE DEHYDROGENASE
+

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

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

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

What substrate does this

inhibitor resemble?

N
R
NAD-OH

NADH

Which plot describes the inhibition of lactate dehydrogenase

by NAD-OH when NADH is the varied substrate?
H+

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

Which plot describes the inhibition of lactate dehydrogenase

by NAD-OH when pyruvate is the varied substrate?
H+

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

Which plot describes the inhibition of lactate dehydrogenase

by oxamate when NADH is the varied substrate?
H+

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

Which plot describes the inhibition of lactate dehydrogenase

by oxamate when pyruvate is the varied substrate?
H+

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

Competitive inhibition seen

if varied S is pyruvate

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

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

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

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:

g lyceraldeh yd e-3-Pi

ORDERED TRI BI MECHANISM

NAD +

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

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

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:

g lyceraldeh yd e-3-Pi

ORDERED TRI BI MECHANISM

NAD +

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

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

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

Example: Product Inhibition

-galactosidase (lactase)
HOCH2

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

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

Cl CH2 C CH NH S

CHY-HIS=N :

CH3

CHY-HIS=N-

CH2

Cl
(X)

irreversibly
inactivated

Suicide Inhibitors: Aspirin

CO2H

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

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

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

Deprenyl
N

Pargyline
N

http://www.csusm.edu/DandB/AD.html

14