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

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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

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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

http://cti.itc.virginia.edu/~cmg/Demo/pdb/cycox/cycox_2.html

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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

+
OH
salicylic acid

Cox-1 CH2O
C CH 3 O

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

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. 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 O

Clorgyline N

Deprenyl N

Pargyline N

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

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