Enzyme Enzyme InhibitionInhibition

ByBy

Prof. V.K. GuptaProf. V.K. Gupta

Department of BiochemistryDepartment of Biochemistry

Kurukshetra University, KurukshetraKurukshetra University, Kurukshetra

email: vkgupta59@rediffmail.com email: vkgupta59@rediffmail.com

Enzyme Enzyme Inhibitor Inhibitor

An Enzyme inhibitor is a compound that An Enzyme inhibitor is a compound that decreases or tends to decrease the rate decreases or tends to decrease the rate of an enzyme catalyzed reaction by of an enzyme catalyzed reaction by influencing the binding of influencing the binding of S S and /or its and /or its turnover number.turnover number.

Type of Enzyme Type of Enzyme Inhibitors Inhibitors

ReversibleReversible

IrreversibleIrreversible

Type of Type of InhibitorsInhibitors

CompetitiveCompetitive

UncompetitiveUncompetitive

Non- CompetitiveNon- Competitive

Active Site Active Site DirectedDirected

Suicide / kSuicide / kcatcat

InhibitorsInhibitors

Inhibitor binds to Enzyme reversibly through weak non-covelent Inhibitor binds to Enzyme reversibly through weak non-covelent

interactionsinteractions

An Equilibrium is established between the free inhibitor & EI Complex An Equilibrium is established between the free inhibitor & EI Complex

and is defined by an equilibrium constant (Ki)and is defined by an equilibrium constant (Ki)

The activity of Enzyme Is fully restored on removing the Inhibitor by The activity of Enzyme Is fully restored on removing the Inhibitor by

dialysis.dialysis.

Reversible Inhibitors depending on concentration of E, S and I, show a Reversible Inhibitors depending on concentration of E, S and I, show a

definite degree of inhibition which is reached fairly rapidly and remains definite degree of inhibition which is reached fairly rapidly and remains

constant when initial velocity studies are carried out.constant when initial velocity studies are carried out.

Reversible InhibitionReversible Inhibition

IEIE +

Irreversible InhibitionIrreversible Inhibition

Inhibitor binds at or near the active site of the enzyme irreversibly, usually Inhibitor binds at or near the active site of the enzyme irreversibly, usually by covalent bonds, so it can’t dissociate from the enzymeby covalent bonds, so it can’t dissociate from the enzyme

No equilibrium exitsNo equilibrium exits

Enzyme activity is not regained on dialysisEnzyme activity is not regained on dialysis

Effectiveness of I is expressed not by equilibrium constant but by a Effectiveness of I is expressed not by equilibrium constant but by a velocity constant, which determines the fraction of the enzyme inhibited in velocity constant, which determines the fraction of the enzyme inhibited in a given period of time by a certain concentration of the Ia given period of time by a certain concentration of the I

EE II EE II++

Competitive Inhibition Competitive Inhibition

A competitive I combines with the free enzyme to form an EI A competitive I combines with the free enzyme to form an EI complex in a manner that prevents S binding complex in a manner that prevents S binding

Binding of S & I is mutually exclusiveBinding of S & I is mutually exclusive

Inhibition can be reversed by increasing the concentration of S at a Inhibition can be reversed by increasing the concentration of S at a constant [I]constant [I]

Degree of inhibition will depend on the concentrations of S & I and Degree of inhibition will depend on the concentrations of S & I and on the relative affinities of the enzyme for S & Ion the relative affinities of the enzyme for S & I

Binding of S & I in different Binding of S & I in different SituationsSituations1.1. Classical Competitive Inhibition (S & I compete for the Classical Competitive Inhibition (S & I compete for the

same binding site)same binding site)

SS II

EnzymeEnzyme

2. S & I are mutually 2. S & I are mutually exclusive because of exclusive because of steric hindrancesteric hindrance

3. S & I have a common 3. S & I have a common binding group on the binding group on the enzyme.enzyme.

EnzymeEnzyme

II

EnzymeEnzyme

II SSSS

4. The binding sites for S & I are distinct but 4. The binding sites for S & I are distinct but overlapping.overlapping.

EnzymeEnzyme

II

SS

5. Binding of I to a distinct inhibitor site causes a 5. Binding of I to a distinct inhibitor site causes a conformational change in the enzyme that distorts conformational change in the enzyme that distorts or masks the S binding site or vice versa.or masks the S binding site or vice versa.

EnzymeEnzyme

II SS

EnzymeEnzyme

II

SS

EnzymeEnzyme

II

SS

Examples for Competitive Inhibition

COO-

CH2

CH2

COO-

+ FADSDH HCCOO-

-OOCCH

+ FADH2

Succinate Fumarate

i)

ii) Cometitive inhibition accounts for the antibacterial action of sulfanilamide Cometitive inhibition accounts for the antibacterial action of sulfanilamide which is a structural analog of PABAwhich is a structural analog of PABA

Sulfanilamide inhibits the bacterial enzyme dihydropteroate synthetase Sulfanilamide inhibits the bacterial enzyme dihydropteroate synthetase which catalyzes the incorporation of PABA into 7,8-dihydropteroic acid.which catalyzes the incorporation of PABA into 7,8-dihydropteroic acid.

H2N COOH

PABA

H2N S NH2

O

O

Sulfanilamide

Malonate is a competitive inhibitor of SDH.Malonate is a competitive inhibitor of SDH.

Derivation of velocity Derivation of velocity equationequation

Ki = Ki = [E] [I][E] [I] [EI][EI]

or [EI]or [EI] = = [E] [I][E] [I] KiKi

In the steady state assumptionIn the steady state assumption

[E] [S][E] [S] [ES][ES]

kk-1-1 + k + k22

kk11

== == KmKm

E + S ES E+P

I

EI + S

+

No ReactionX

Ki

k1 k2

k-1

[ES][ES] = = [E] [S][E] [S] KmKm

v=kv=k22[ES] [ES] Vmax = k Vmax = k2 2 [E][E]T T Now [E]Now [E]T T = [E] + [ES] + [EI]= [E] + [ES] + [EI]

X

kk22 [ES] [ES]

kk22 ( [E] + [ES] + [EI] ) ( [E] + [ES] + [EI] )

VVmax max = k= k2 2 ( [E] + [ES] + [EI] )( [E] + [ES] + [EI] )

[ES] [ES]

[E] + [ES] + [EI] [E] + [ES] + [EI] == ==

vvVVmaxmax

Putting the value of [ES] and [EI}Putting the value of [ES] and [EI}

[E] [S][E] [S] KmKm

[E] [E] [S] [E] [I][E] [E] [S] [E] [I] Km KKm Kii

++ ++

vvVVmaxmax

==

[S][S] KmKm

[S] [I][S] [I] Km KKm Kii

1 +1 + ++

==vvVVmaxmax

[S][S]

[I] [I] KiKiKKmm + [S] + + [S] + KKmm

==vvVVmaxmax

[S][S]

[I] [I] KiKiKKmm (1+ ) (1+ )+ [S]+ [S]

==vvVVmaxmax

Multiplying by kMultiplying by km m both in the numerator and the both in the numerator and the

denominator denominator

In the presence of a competitive inhibitor KIn the presence of a competitive inhibitor Km m increases increases

VVmax max unchanged unchanged

==vvVVmaxmax

[S][S]

KmKmappapp + [S] + [S]

Where KmWhere Kmappapp

[I] [I] KiKi==

No inhibitorNo inhibitor

+ C Inhibitor+ C Inhibitor

VVmaxmax

½ V½ Vmaxmax

KKmm KmKmappapp[s][s]

vv

KKmm (1+ ) (1+ )==

Lineweaver Burk plot KmVmax

=1v

[I]2

[I]1

1

Kmapp

1

Km

( 1+ )[I]Ki 1

[S] 1Vmax

+

Slope = Km

Vmax ( 1+

)[I]Ki

Calculation of KCalculation of Kii

From slope of the double reciprocal plot in the presence of a C. From slope of the double reciprocal plot in the presence of a C. Inhibitor which is egual toInhibitor which is egual to

From KmFrom Kmapp app which is given by which is given by

[I] [I] KiKiKmKmappapp KKmm (1+ ) (1+ )==

(1+ )(1+ ) [I] [I] KiKi

KmKmVVmaxmax

Slope =Slope =

KKmm

VVmaxmax

KKmm

VVmax max KKii

Slope =Slope =

- K- Kii

[I][I]

A graphical method is preferred to direct substitution of A graphical method is preferred to direct substitution of numbers to allow errors in individual determination to be numbers to allow errors in individual determination to be

averaged out averaged out

KmKmappapp

VVmaxmax

From the replot of slope vs. [I] From the replot of slope vs. [I]

Slope =Slope =KKmm

VVmax max

++KKmm

VVmaxmaxKiKi

[I][I]

Slope =Slope =

KKmm

KKmm

KKii

Slope =Slope =

- K- Kii

[I][I]

KmKmappapp

From replot of KmFrom replot of Kmapp app Vs. [I]Vs. [I]

KKmm

KKii

[I][I]++KKmmKmKmappapp = =

KK mm

VV max max [S

] K[S] K ii

Slope =Slope =

- K- Kii

[I][I]

(1+ )(1+ ) Km Km [S][S]

11VVmaxmax

11

vv

11VVmaxmax

Slope = 0Slope = 0[S] = [S] =

[S][S]22

[S][S]11

Increasing [S]

Increasing [S]

(1+ )(1+ ) [S] [S] KmKm

From Dixon’s plotFrom Dixon’s plot KmKmVmax[S] Ki Vmax[S] Ki

==11vv

11Vmax Vmax

[I] [I] ++ (1+ )(1+ ) Km Km

[S][S]

Non-competitive Inhibition Non-competitive Inhibition

An inhibitor that binds to an enzyme to form a dead end complex, An inhibitor that binds to an enzyme to form a dead end complex,

whether or not the active site is occupied by a substrate is termed as a whether or not the active site is occupied by a substrate is termed as a

NC InhibitorNC Inhibitor

Can bind either to E or ES complexCan bind either to E or ES complex

Since I doesn't bear structural resemblance to the S, it must bind to the Since I doesn't bear structural resemblance to the S, it must bind to the

enzyme at a site distinct from the S binding siteenzyme at a site distinct from the S binding site

The presence of I does not affect S bonding but does interfere with the The presence of I does not affect S bonding but does interfere with the

catalytic functioning of the enzyme catalytic functioning of the enzyme

The binding of I often deforms the E so that it doesn’t form ES complex The binding of I often deforms the E so that it doesn’t form ES complex

at a normal rate and once formed, ES complex doesn’t decompose at at a normal rate and once formed, ES complex doesn’t decompose at

normal rate to yield productsnormal rate to yield products

A NC I doesn’t affect the Km because the binding of I does not A NC I doesn’t affect the Km because the binding of I does not

block S binding or vice-versablock S binding or vice-versa

I effectively lowers the concentration of active enzyme and I effectively lowers the concentration of active enzyme and

hence decreases the apparent Vhence decreases the apparent Vmaxmax

since there is no competition between S & I, the inhibition is not since there is no competition between S & I, the inhibition is not

reversed by increasing the [S]reversed by increasing the [S]

EnzymeEnzyme EnzymeEnzyme

EnzymeEnzyme EnzymeEnzyme

SS

IISS

II

Examples for Non- Examples for Non- Competitive InhibitionCompetitive Inhibition

1.1. Enzymes requiring divalent metal ions (e.g. MgEnzymes requiring divalent metal ions (e.g. Mg2+ 2+ & Ca& Ca2+ 2+ etc) for their activity are inhibited etc) for their activity are inhibited non-competitively by chelating agents like EDTA which removes metal ions from the non-competitively by chelating agents like EDTA which removes metal ions from the enzymeenzyme

2.2. Enzymes with -SH groups that participate in the maintenance of the three dimensional Enzymes with -SH groups that participate in the maintenance of the three dimensional conformation of the molecule are non-competitively inhibited by heavy metal ions.conformation of the molecule are non-competitively inhibited by heavy metal ions.

EE SH + HgSH + Hg2+2+ EE S HgS Hg+ + + H+ H++

E + S ES E+P

I

EI + S

+

Ki

k2

Ks

+I

ESI

Ks

Ki

Ks = Ks = [E] [S][E] [S] [ES][ES]

[EI] [S][EI] [S] [ES][ES]==

Replacing Ks with KmReplacing Ks with Km

[ES] = [ES] = [E] [S][E] [S] KmKm`̀

No inhibitorNo inhibitor

+ NC Inhibitor+ NC Inhibitor

VVmaxmax

½ V½ Vmaxmax

KKmm [s][s]

vv

½ V½ Vmax imax i

VVmax imax i

Vmax = Decreases.Vmax = Decreases.

Km = UnchangedKm = Unchanged

Lineweaver – Burk PlotLineweaver – Burk Plot

KmKmVmaxi Vmaxi

==11vv

11[S] [S]

11Vmaxi Vmaxi

++

[I][I]22

[I][I]11

No InhibitorNo Inhibitor

KKmm

VVmaxmax

Slope =Slope =

Intercept = Intercept = 11

VVmaxmax

Intercept = Intercept = 11

VVmaximaxi

11

KmKm

1/[s]1/[s]

KK mm

VV max

im

axi

Slop

e =

Slop

e =

Both slope & Both slope & Intercept Intercept

Increased By Increased By the factorthe factor

(1+[ I ] )(1+[ I ] ) KiKi

1/v1/v

KKmm

VmaxVmax- K- Kii

[I][I]

SlopeSlope

[I][I]++KKmm

VmaxVmaxSlope =Slope =

Calculation of KCalculation of Kii

i)i) From the slope of the reciprocal plotFrom the slope of the reciprocal plot

ii)ii) from the intercept of the reciprocal from the intercept of the reciprocal plotplot

iii)iii) from replot of slope of the reciprocal from replot of slope of the reciprocal plot vs [ I ]plot vs [ I ]

KKmm

Vmax KiVmax Ki

In partial NC inhibition In partial NC inhibition this plot is hyperbolicthis plot is hyperbolic

iv.iv. Replot of intercept of the primary plot in the presence Replot of intercept of the primary plot in the presence of a NC I vs [I] is linearof a NC I vs [I] is linear

11

VmaxVmax- K- Kii

[I][I]

InterceptIntercept

In partial NC inhibition In partial NC inhibition this plot is hyperbolicthis plot is hyperbolic

[I][I]++

11

VmaxVmaxIntercept =Intercept =

11

Vmax KiVmax Ki

v.v. Dixon’s PlotDixon’s Plot

KmKm

Vmax [S]Vmax [S]- K- Kii

[I][I]

1/v1/v

[S][S]11

[S][S]22

Intercept = Intercept = (( + + ) )

11

VmaxVmax

KmKm

Vmax [S]Vmax [S]Slope = Slope = (( + + ) )

11

VmaxVmax

11

KiKi

A plot of 1/v vs [I] will be linear at A plot of 1/v vs [I] will be linear at

fixed [E] and [S] for NC inhibitionfixed [E] and [S] for NC inhibition

Uncompetitive Inhibition Uncompetitive Inhibition I doesn't bind to the free E rather it binds to the ES complexI doesn't bind to the free E rather it binds to the ES complex

the binding of an UC I is presumed to cause structural distortion the binding of an UC I is presumed to cause structural distortion of the active site making the enzyme catalytically inactiveof the active site making the enzyme catalytically inactive

the binding of S could cause a conformational change in the E the binding of S could cause a conformational change in the E thereby revealing an I binding sitethereby revealing an I binding site

Inhibition can’t be reversed by increasing the [S] since I doesn't Inhibition can’t be reversed by increasing the [S] since I doesn't compete with S for the same binding site compete with S for the same binding site

EnzymeEnzymeEnzymeEnzyme

SS

EnzymeEnzyme

II SS

UC Inhibition is rare in single-substrate reactions. UC Inhibition is rare in single-substrate reactions.

for e.g. Inhibition of intestinal alkaline phosphatase by L- for e.g. Inhibition of intestinal alkaline phosphatase by L-

phenylalanine. It is common in multisubstrate reactionsphenylalanine. It is common in multisubstrate reactions

E + S E S E + PE + S E S E + P

++

II

ESIESI

[ES] = [ES] = [E] [S][E] [S] KmKm

[ESI] = [ESI] = [E] [S] [I][E] [S] [I] Km KiKm Ki

The equilibria show that at any [I] an infinitely high [S] will not drive all the enzyme to ES form; some non productive ESI complex will always be present. Consequently an UC I will decrease the Vmax

An UC I will also decrease the Kmapp because the reaction

ES + I ESI removes some ES causing the reaction

E + S ES to proceed to the right

No inhibitor

+ UC Inhibitor

Vmax

½ Vmax

Km [s]

½ Vmax i

Vmax i

Vmax = Decreases

Km = Decreases

Kmapp

v

v

Vmax

(1+ )

=

[I]

Ki

[s]

Km

(1+ )[I]

Ki

+[s]

v

Vmaxi=

[s]

Kmapp +[s]

The equation can also be written asThe equation can also be written as

Where Where Vmaxi =Vmax

(1+ )[I]

Ki

Kmapp=Km

(1+ )[I]

Ki

[I]2[I]1

No I

-1/Kmapp-1/Km

Km

VmaxSlope =

1/Vmax

1/Vmaxi

1/v

1/[s]

Increasing [I]

Lineweaver Burk plot

1

v

Km 1 1

Vmax [S] Vmax= +

(1+ )[I]

Ki

Slope remains Unchanged &

Intercept Increases By

the factor

(1+[ I ] ) Ki

Incase of UC Inhibition Ki is that concn of I which halves the value of both Vmax and Km

Calculation of Ki

i) From the slope of the reciprocal plot

ii) From the Km app

iii) From replot of 1/Vmaxi vs [ I ]

1

Vmax- Ki

[I]

1/Vmaxi

1

VmaxKiSlope =

1

Vmaxi

1

Vmax =

(1+ )[I]

Ki

1

Vmaxi

1

Vmax =

1

VmaxKi +

[I]

iv. From replot of 1/Km appvs [I]

1

Km app

1

Km =

(1+ )[I]

Ki

1

Kmapp

1

Km =

1

KmKi +

[I]

1

Km

[I]

1/Kmapp

1

KmKiSlope =

- Ki

iv.iv. Dixon’s PlotDixon’s Plot

11

vv

Km [I] 1Km [I] 1

Vmax Ki VmaxVmax Ki Vmax== ++

(1+ )(1+ )KmKm

[S][S]

The equation for Dixon’s plot is The equation for Dixon’s plot is

-Ki-Ki

1/Vmax

1

Vmaxi

1/v

[I]

(1+ )Km

[S]

(1+ )Km

[S]

[S] =

∞

1

VmaxKi

Slope =Increasing [S

]

Irreversible InhibitionIrreversible Inhibition

An irreversible Inhibitor binds at or near the active site of the An irreversible Inhibitor binds at or near the active site of the

enzyme irreversibly, usually by covalent bonds, so that it can’t enzyme irreversibly, usually by covalent bonds, so that it can’t

subsequently dissociate from the enzymesubsequently dissociate from the enzyme

The I destroys as essential functional group on the enzyme that The I destroys as essential functional group on the enzyme that

participates in normal S binding or catalytic action. As a result the participates in normal S binding or catalytic action. As a result the

enzyme is rendered permanently inactiveenzyme is rendered permanently inactive

Compounds which irreversibly denature the enzyme protein or Compounds which irreversibly denature the enzyme protein or

cause non-specific inactivation of the active site are not usually cause non-specific inactivation of the active site are not usually

regarded as irreversible inhibitors.regarded as irreversible inhibitors.

Examples:Examples:

Organophosphorus compounds (such as DFP) irreversibly react with the Organophosphorus compounds (such as DFP) irreversibly react with the

–OH group of essential serine residue of some enzymes–OH group of essential serine residue of some enzymes

DFP (Diisopropylphosphofluoridate) is a nerve poison since it inactivates DFP (Diisopropylphosphofluoridate) is a nerve poison since it inactivates

acetylcholinesterase that plays an important role in the transmission of acetylcholinesterase that plays an important role in the transmission of

nerve impulses. nerve impulses.

EE CHCH22-OH + F—P=O -OH + F—P=O

EE CHCH22-O- F—P=O + HF -O- F—P=O + HF

OCH(CHOCH(CH33))22

OCH(CHOCH(CH33))22

OCH(CHOCH(CH33))22

OCH(CHOCH(CH33))22

DFPDFP Catalytically inactive Catalytically inactive enzymeenzyme

(Con

trol

) no

Inhi

bito

r

(Con

trol

) no

Inhi

bito

r+

NC Inhib

itor

+ NC In

hibito

rIr

reve

rsib

le I

nhib

itor

Irre

vers

ible

Inh

ibito

r

[E][E]ii

[E][E]TT

VmaxVmax

To distinguish between irreversible & NC To distinguish between irreversible & NC InhibitionInhibition

Irreversible inhibitors

Active site directed irreversible Inhibitors

or

(Affinity labels)

Suicide Inhibitors

(Mechanism-based Inhibitors)

or

(kcat Inhibitors)

Types of Irreversible Inhibitors

Affinity labelsAffinity labels

An affinity label is a chemically reactive compound that An affinity label is a chemically reactive compound that

is designed to resemble the substrate of an enzyme so is designed to resemble the substrate of an enzyme so

that it binds at the active site and forms a stable that it binds at the active site and forms a stable

covalent bond with a susceptible group of the nearby covalent bond with a susceptible group of the nearby

residue in the enzyme protein.residue in the enzyme protein.

Affinity labels are very useful for identifying catalytically Affinity labels are very useful for identifying catalytically

important residuesimportant residues

Examples:

TPCK acts as an affinity label for Chymotrypsin; even at very low concn TPCK quantitatively inactivates chymotrypsin; TPCK is identical in structure to a substrate of this enzyme i.e. tosyl-L-phenylalanyl methyl ester, except that the carboxylic ester is replaced by the chloromethyl group.

NH

O CH2Cl

S

O

O

NH

O OCH3

S

O

O

tosyl-L-phenylalanine methyl esterTPCK

(Affinity label) (Substrate)

NH

O CH2

S

O

OCl

NN H

CH2 His 57

NH

O CH2

S

O

O

NN H

CH2 His 57Cl- + H+

Alkylated derivative of His 57

(inactive Enzyme)

O

HO

phenylpropionate

Excess concn of this prevent the inactivation by TPCK

TPCK is attacked in a nucleophilic reaction by the N atom of the imidazole side chain of His57. the binding of TPCK to the Enz Brings the reactive –Cl group in close proximity to the His57 residue and facilitates the formation of a covelent bond between the I & imidazole side chain

CH3

CH3

Suicide InhibitorsSuicide Inhibitors

A suicide inhibitor is a relatively inert molecule that is transformed by an A suicide inhibitor is a relatively inert molecule that is transformed by an enzyme at its active site into a reactive compound that irreversibly enzyme at its active site into a reactive compound that irreversibly inactivates the enzymeinactivates the enzyme

They are substrate analogs designed so that via normal catalytic action of They are substrate analogs designed so that via normal catalytic action of the enzyme, a very reactive group is generated. the enzyme, a very reactive group is generated.

The latter forms a covalent bond with a nearby functional group within the The latter forms a covalent bond with a nearby functional group within the active site of the enzyme causing irreversible inhibition.active site of the enzyme causing irreversible inhibition.

Such inhibitors are called suicide inhibitors because the enzyme appears Such inhibitors are called suicide inhibitors because the enzyme appears to commit suicide.to commit suicide.

e.g. FdUMP is a suicide inhibitor of thymidylate synthase.e.g. FdUMP is a suicide inhibitor of thymidylate synthase.

During thymidylate synthesis, NDuring thymidylate synthesis, N55,N,N1010- methyleneTHF is - methyleneTHF is converted to 7,8-dihydrofolate; methyleneTHF is regenerated converted to 7,8-dihydrofolate; methyleneTHF is regenerated in two stepsin two steps

Conversion of dUMP to dTMP and its inhibition by FdUMPConversion of dUMP to dTMP and its inhibition by FdUMP

For understanding the regulation of enzyme activity within the living cells

To elucidate the kinetic mechanism of an enzyme catalyzing a multisubstrate reaction

Useful in elucidating the cellular metabolic pathways by causing accumulation of intermediates

Indentifiction of the catalytic groups at the active site

Provide information about substrate specificity of the enzyme

Importance of Enzyme Inhibition

Form the basis of drug designing. The whole area of selective toxicity , including the use of antibiotic, toxin, insecticides etc is based on the exploitation of species differences in the susceptibility to enzyme inhibitors.

Competitive inhibitors are useful in x-rays crystallographic studies to pin point the active site in crystal structure and thus revealing how the surrounding amino acid residues interact with the bound molecule.

To treat methanol poisoning