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
-Ic binds to free E at active site where S binds Km
+
-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 y-int = 1/Vmax -I c

Vo

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

-Inc is not structurally similar to S; is not an S E + S ES P + E

Km
-Inc binds to free E or ES at a site where S + +
does not bind I nc I nc
-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 slope = Km . (1+ [Inc ] / Kc ) / Vmax

+I
+ I nc
Vo 1 / Vo
-I

So

y-int = (1+[I nc] / Knc ) / Vmax 1 / So

slope = K m /Vmax
x-int = -1/Km
y-int = 1/Vmax

Examples: Competitive and Noncompetitive Inhibition

H+
O H H H O
O OH
C C + CH3 C CO2-
H2N + CH3 C CO2- NH2
H
N N
pyruvate L-lactate
R R
**NADH
(reduced form) **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 H O
O OH
H2N C C + CH3 C CO2-
+ CH3 C CO2- NH2
H
N N
pyruvate L-lactate
R R
**NADH **NAD
(reduced form)
(oxidized form)

HO H O
O O
C NH
INHIBITORS: 2 H2N C CO2- CH3CH2HN C CO2-
N 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 H O
O OH
H2N C C + CH3 C CO2-
+ CH3 C CO2- NH2
H
N N
pyruvate L-lactate
R R
**NADH **NAD
(reduced form)
(oxidized form)

HO H O
C NH What substrate does this
INHIBITORS: 2 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+ ORDERED BI BI MECHANISM
O H H H O
O OH NADH PYR LAC NAD+
HNC C + CH3 C CO2-
2 + CH3 C CO2- NH2 S1 S2 P1 P2
H
N N
pyruvate L-lactate
R R
**NADH HO H O **NAD E ES1 ES1S2 EP1P2 EP2 E
(reduced form)
C (oxidized form)
NH2

N
Competitive inhibition
R
NAD-OH seen if varied S is NADH
Noncompetitive Competitive
+I +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+ ORDERED BI BI MECHANISM
O H H H O
O OH NADH PYR LAC NAD+
HNC C + CH3 C CO2-
2 + CH3 C CO2- NH2 S1 S2 P1 P2
H
N N
pyruvate L-lactate
R R
**NADH HO H O **NAD E ES1 ES1S2 EP1P2 EP2 E
(reduced form)
C (oxidized form)
NH2

N
Noncompetitive inhibition
R
NAD-OH seen if varied S is pyruvate
Noncompetitive Competitive
+I +I
1 / Vo 1 / Vo

-I -I

1/PYRUVATE 1/PYRUVATE

5
 Which plot describes the inhibition of lactate dehydrogenase
by oxamate when NADH is the varied substrate?
H+ ORDERED BI BI MECHANISM
O H H H O
O OH NADH PYR LAC NAD+
HNC C + CH3 C CO2-
2 + CH3 C CO2- NH2 S1 S2 P1 P2
H
N N
pyruvate L-lactate
R R
**NADH O EP2
**NAD E ES1 ES1S2 EP1P2 E
(reduced form)
H2N C CO2- (oxidized form)

oxamate
Noncompetitive inhibition
seen if varied S is NADH
Noncompetitive Competitive
+I +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+ ORDERED BI BI MECHANISM
O H H H O
O OH NADH PYR LAC NAD+
HNC C + CH3 C CO2-
2 + CH3 C CO2- NH2 S1 S2 P1 P2
H
N N
pyruvate L-lactate
R R
**NADH O EP2
**NAD E ES1 ES1S2 EP1P2 E
(reduced form)
H2N C CO2- (oxidized form)

oxamate
Competitive inhibition seen
if varied S is pyruvate
Noncompetitive Competitive
+I +I
1 / Vo 1 / Vo

-I -I

1/PYRUVATE 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

UNCOMPETITIVE Equilibria Scheme

-Iu is not structurally similar to S; is not an S E + S ES P+ E
-Iu binds to ES only; S opens up a site for Km
u I +
-Iu 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 +I
1/Vo

- Iu -I
Vo
slope = Km / Vmax

So 1 / So
x-int = -(1+ [Iu] / Ku ) / Km
y-int = 1 / Vmax
x-int = -1 / Km

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 O
O H H O
C C H O -
NH2 C O P-O
+ H C OH + HO P O- H2N C O
+ H C OH
N CH2OPi OH N CH2OPi
R g lyceraldeh yd e-3-Pi R
**NAD 1,3-b isphospho glycer ate
**NADH
(oxid ized fo rm )
(red uce d fo rm)
O
INHIBITOR: HO As O-
ORDERED TRI BI MECHANISM
OH NAD + GAP Pi 1,3-BPG N ADH

S1 S2 S3 P1 P2

E ES 1 ES' 1 P 2 ES' 1 P 2 EP 1 P 2 EP 2 E

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 O
O H H O
C C H O -
NH2 C O P-O
+ H C OH + HO P O- H2N C O
+ H C OH
N CH2OPi OH N CH2OPi
R g lyceraldeh yd e-3-Pi R
**NAD 1,3-b isphospho glycer ate
**NADH
(oxid ized fo rm )
(red uce d fo rm)
O
INHIBITOR: HO As O-
ORDERED TRI BI MECHANISM
OH NAD + GAP Pi 1,3-BPG N ADH

S1 S2 S3 P1 P2

E ES 1 ES' 1 P 2 ES' 1 P 2 EP 1 P 2 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 O

NAD + GAP Pi O 1,3-BPG NADH
HO As O-
S1 S 2 S 3 HO P O- P1 P2 OH
OH

E ES1 ES'1P2 ES' 1P2 EP1P2 EP2 E

.
Noncompetitive Competitive Uncompetitive
+I +I
1 / Vo 1 / Vo 1 / Vo +I

-I -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 P+ E
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 1 / So
x-int = -1 / Km

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 Example: Product Inhibition
β -galactosidase (lactase)
HOCH2
lactose
O
HOCH2 OH
O O OH
HO
OH OH
O
OH H H

HOCH2 HOCH2
HO O OH O
OH OH
HO OH
OH OH
galactose glucose

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
Vo +I -I

So
1 / So

Michaelis-Menten and Lineweaver-Burk plots “look” noncompetitive

10
 Suicide Inhibition
+I
1 / Vo
-I
Vo +I -I

So
1 / So
k1
k2
E + S ES P + E
k -1
Nu:
+
R-X ( = I) The suicide inhibitor removes E so that the
[ES] is lower, Vmax is lower, and inhibition
X
E
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
k2
E + S ES 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
X -I lowers [E], therefore lowering [ES] and Vo
E -I lowers maximum amount of [ES] because it removes E
-V o can never reach Vmax
Nu -resembles noncompetitive inhibition because inhibition cannot
be relieved at high S
R
inactived enzyme

O O
CHY-HIS=N : Cl CH2 C CH NH S CH3 CHY-HIS=N- R
CH2 O
irreversibly
Cl inactivated
(X)

Suicide Inhibitors: Aspirin

CO2H
Drug Target Enzymes
(Box 21-1)
O C CH 3
Aspirin O Cyclooxygenases 1 and 2
aspirin
O
C CH 3
CO2H CO2Na
NH
CH CH3 CH CH3

"Simple"
Reversible
Inhibitors
CH2 O OH
CH CH3 tylenol
CH3 CH3 O
CH3 CNHCHCO2 -
ibuprofen naproxen CH2
N-acetylcysteine
antidote for SH
http://cti.itc.virginia.edu/~cmg/Demo/pdb/cycox/cycox_2.html 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 CH2O
salicylic acid C CH 3
O

http://www.scripps.edu/pub/goodsell/pdb/pdb17/pdb17_1.html

Suicide Inhibitors: New NSAIDs

Drug Target Enzyme
Celebrex 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)

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 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

Pargyline

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

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