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LECTURE 4:LECTURE 4:Reaction Mechanism and InhibitorsReaction Mechanism and Inhibitors

Kinetic data cannot unambiguously establish a reaction mechanism.Although a phenomenological description can be obtained the nature of the reaction intermediates remain indeterminate and other independent

measurements are needed.

Modes of Antimicrobial ActionModes of Antimicrobial ActionEnzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n and

Des

cripti

on

[II] binds to free [E] only,and competes with [S];increasing [S] overcomesInhibition by [II].

[II] binds to free [E] or [ES] complex; Increasing [S] cannot overcome [II] inhibition.

[II] binds to [ES] complex only, increasing [S] favorsthe inhibition by [II].

E + S→ES→E + P+II↓EII

←

↑

E + S→ES→E + P+ +II II↓ ↓EII+S→EIIS

←

↑ ↑

E + S→ES→E + P+II

↓EIIS

←

↑

EI

S

Juang RH (2004) BCbasics

LECTURE LAYOUTLECTURE LAYOUTREACTION MECHANISMSREACTION MECHANISMSA.A. Sequential ReactionsSequential ReactionsB.B. Random Random BisubstrateBisubstrate ReactionsReactionsC.C. PingPing--Pong ReactionsPong ReactionsINHIBITORSINHIBITORS1.1. Irreversible Irreversible 2.2. ReversibleReversible

Competitive inhibition,Competitive inhibition, Uncompetitive inhibition.Uncompetitive inhibition. Noncompetitive inhibition Noncompetitive inhibition

INTRODUCTIONINTRODUCTION

Thus far, we have considered only the simple Thus far, we have considered only the simple case of enzymes that act upon a single case of enzymes that act upon a single substrate, substrate, SS. This situation is not common. . This situation is not common. Usually, enzymes catalyze reactions in which Usually, enzymes catalyze reactions in which two (or even more) substrates take part.two (or even more) substrates take part.

Consider the case of an enzyme catalyzing a Consider the case of an enzyme catalyzing a reaction involving two substrates, reaction involving two substrates, AA and and BB, and , and yielding the products yielding the products PP and and QQ::

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Reaction MechanismsReaction MechanismsA: Sequential ReactionsA: Sequential Reactions All substrates must combine with enzyme All substrates must combine with enzyme

before reaction can occurbefore reaction can occur

Bisubstrate reactions

B. Random Bisubstrate Reactions Group transfer reactionsGroup transfer reactions One or more products One or more products

released before all released before all substrates addedsubstrates added

C. Ping-Pong Reactions

QUIZ (10 min)QUIZ (10 min)1.1. How is enzyme specificity achieved ?How is enzyme specificity achieved ?2.2. Calculate Vmax & KM from the following data, and dCalculate Vmax & KM from the following data, and does the oes the

reaction obey Michaelisreaction obey Michaelis--Menten kinetics ?Menten kinetics ?

[DNA]mol total

nucleotides/L

Free nucleotides in solution,V (pmol/L)

0 min 10 min1.0 x 10-5 0.05 5.11.0 x 10-6 0.04 4.51.0 x 10-7 0.06 3.21.0 x 10-8 0.04 1.41.0 x 10-9 0.04 0.23

ANSWERSANSWERS1.1. The enzyme specificity is achieved The enzyme specificity is achieved

through the characteristic of active sitethrough the characteristic of active site2.2. Vmax = Vmax = 4.366954.36695

KM = KM = 2.2E2.2E--0808RR22 = = 0.999864, so the reaction obeys 0.999864, so the reaction obeys MichaelisMichaelis--Menten kinetics Menten kinetics

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An important number of compounds have An important number of compounds have the the ability to combine with certain enzymesability to combine with certain enzymes in either a in either a reversible or irreversible manner, and thereby reversible or irreversible manner, and thereby block catalysis by that enzymeblock catalysis by that enzyme

Such compounds are called Such compounds are called INHIBITORS INHIBITORS and and include drugs, include drugs, antibioticsantibiotics, , poisonspoisons, anti , anti metabolitesmetabolites, as well as , as well as products of enzymic products of enzymic reactionsreactions

Two general classes of inhibitors are recognized ; Two general classes of inhibitors are recognized ; Irreversible Irreversible ReversibleReversible

INHIBITORS Inhibitors of Michaelis-Menten Enzymes

An irreversible inhibitor forms a covalent bond An irreversible inhibitor forms a covalent bond with a with a specific function, usually an amino acid residue, specific function, usually an amino acid residue, which may, in some manner, be associated with the which may, in some manner, be associated with the catalytic activity of the enzymecatalytic activity of the enzyme

There are many examples of enzyme inhibitors which There are many examples of enzyme inhibitors which covalently bind not at the active site, but physically covalently bind not at the active site, but physically block the active siteblock the active site

The inhibitor cannot be released by dilution or The inhibitor cannot be released by dilution or dialysisdialysis; ; kinetically, the concentration and hence the kinetically, the concentration and hence the velocity of active enzyme is lowered in proportion to velocity of active enzyme is lowered in proportion to the concentration of the inhibitor and thus the effect the concentration of the inhibitor and thus the effect is that of noncompetitive inhibition: is that of noncompetitive inhibition:

1. IRREVERSIBLE INHIBITORS

Irreversible Inhibition E + S ES E + P + I EI

KS

K I

Examples of irreversible inhibitors include;Examples of irreversible inhibitors include; diisopropyl fluorophosphate diisopropyl fluorophosphate which reacts irreversibly withwhich reacts irreversibly with serine serine

proteases, proteases, chymotrypsin, chymotrypsin, andand iodoacetate iodoacetate which reacts with essential sulfhydryl group of an which reacts with essential sulfhydryl group of an

enzyme such as enzyme such as triose phosphate dehydrogenasetriose phosphate dehydrogenase: :

E-SH+ICH2COOH E-SCH2COOH+HI

Reaction of the irreversible inhibitor diisopropylfluorophosphate (DFP) with a serine protease

Diisopropyl fluorophosphate

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A unique type of irreversible inhibition has been recently A unique type of irreversible inhibition has been recently described as kdescribed as kcatcat inhibition in that a latent inhibitor is inhibition in that a latent inhibitor is activated to an active inhibitor by binding to the active activated to an active inhibitor by binding to the active site of the enzyme.site of the enzyme.

The newly generated inhibitor now reacts chemically with The newly generated inhibitor now reacts chemically with the enzyme leading to its irreversible inhibitionthe enzyme leading to its irreversible inhibition

These inhibitors have great potential as These inhibitors have great potential as drugsdrugs in highly in highly specific probes for active sites since they are not specific probes for active sites since they are not converted from the latent to the active form except by converted from the latent to the active form except by their specific target enzymestheir specific target enzymes

An excellent example is the inhibition of An excellent example is the inhibition of DD--33--hydroxyl hydroxyl decanoyl ACP clehydrasedecanoyl ACP clehydrase (of E. coli) by the latent inhibitor (of E. coli) by the latent inhibitor 33--decynoyldecynoyl--NN--acetyl cystamineacetyl cystamine according to the following according to the following sequences of events:sequences of events:

2. REVERSIBLE INHIBITION2. REVERSIBLE INHIBITION As the term implies, this type of inhibition involves As the term implies, this type of inhibition involves

equilibrium between the enzyme and the inhibitor, equilibrium between the enzyme and the inhibitor, the equilibrium constant (Ki) being a measure of the equilibrium constant (Ki) being a measure of the affinity of the inhibitor for the enzyme. the affinity of the inhibitor for the enzyme.

Three distinct types of reversible inhibition are Three distinct types of reversible inhibition are known; known; Competitive inhibition,Competitive inhibition, Uncompetitive inhibition Uncompetitive inhibition Noncompetitive inhibition Noncompetitive inhibition

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A. Competitive InhibitionA. Competitive Inhibition Compounds that may or may not be structurally Compounds that may or may not be structurally

related to the natural substrate combine related to the natural substrate combine reversibly with the enzyme at or near the active reversibly with the enzyme at or near the active sitesite

The inhibitor and the substrate therefore The inhibitor and the substrate therefore compete for the same site according to the compete for the same site according to the reaction: reaction:

]S[K

]I[1K

]S[VV

IM

max

Competitive Inhibition

Succinate Glutarate Malonate Oxalate

Succinate Dehydrogenase

Substrate Competitive InhibitorProduct

Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.49

C-OO-

C-H

C-H

C-OO-

C-OO-

H-C-H

H-C-H

C-OO-

C-OO-

H-C-H

H-C-H

H-C-H

C-OO-

C-OO-

C-OO-

C-OO-

H-C-H

C-OO-

ES and EI complexes are formed, but EIS complexes are never produced. One can conclude that high concentrations of substrate will overcome the inhibition by causing the reaction sequence to swing to the right. The velocity of reaction can be calculated by the following equation

C o m p etitiv e in h ib ito r

-I

+I

-1/KM -1/[K M(1+1/KI)]

1/V

1/S

Among other enzymes that may undergo competitive Among other enzymes that may undergo competitive inhibition (Table 1) is inhibition (Table 1) is succinic dehydrogenasesuccinic dehydrogenase, which , which readily oxidizes succinic acid to fumaric acid. readily oxidizes succinic acid to fumaric acid.

If increasing concentrations of malonic acid, which closely If increasing concentrations of malonic acid, which closely resembles succinic acid in structure, are added, however, resembles succinic acid in structure, are added, however, succinic dehydrogenase activity falls succinic dehydrogenase activity falls markedly. This markedly. This inhibition can now be reversed by increasing in turn the inhibition can now be reversed by increasing in turn the concentration of the substrate succinic acid. concentration of the substrate succinic acid.

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HIV protease vs Aspartyl protease

Asymmetricmonomer

↓ HIV protease HIV protease (homodimer)HIV Protease inhibitor is used in treating AIDS

Symmetricdimer

Asp

subunit 2

↑Aspartyl protease (monomer)

subunit 1Asp

domain 1 domain 2

Asp Asp

Juang RH (2004) BCbasics

B. Uncompetitive InhibitionB. Uncompetitive Inhibition Compounds that combine only with the ES complex, not Compounds that combine only with the ES complex, not

with the free enzyme, are called uncompetitive inhibitors. with the free enzyme, are called uncompetitive inhibitors. The inhibition is not overcome by high substrate The inhibition is not overcome by high substrate concentrations. concentrations.

The structure of the protease is The structure of the protease is shown by the red, blue and yellow shown by the red, blue and yellow ribbons. The inhibitor is shown as ribbons. The inhibitor is shown as the smaller ballthe smaller ball--andand--stick structure stick structure near the centre. Created from PDBnear the centre. Created from PDB

Human immunodeficiency virusScanning electron micrograph of HIV-1 (in green) budding from cultured lymphocyte. Multiple round bumps on cell surface represent sites of assembly and budding of virions

Peptide-based protease inhibitor ritonavir

HIV protease HIV protease in a complex with the protease inhibitor in a complex with the protease inhibitor ritonavirritonavir

KKMM value is consistently smaller than the Kvalue is consistently smaller than the KMM value value of the uninhibited reaction which implies that S is of the uninhibited reaction which implies that S is more effectively bound to the enzyme in the more effectively bound to the enzyme in the presence of the inhibitor. presence of the inhibitor.

The equation used to calculate the velocity of the The equation used to calculate the velocity of the noncompetitive inhibition is as follows noncompetitive inhibition is as follows

IM

max

K]I[1]S[K

]S[VV

Uncompetitive inhibitor

-I

+I

-1/V m ax

(1+[I]/K I)/Vm ax

-(1+[I]/K I)/K M

-1/K M

1/S

1/V

C. Noncompetitive InhibitionC. Noncompetitive Inhibition Compounds that reversibly bind with either the enzyme or Compounds that reversibly bind with either the enzyme or

the enzyme substrate complex are designated as the enzyme substrate complex are designated as noncompetitive inhibitorsnoncompetitive inhibitors

Noncompetitive inhibition therefore differs from Noncompetitive inhibition therefore differs from competitive inhibition in that the inhibitor can combine competitive inhibition in that the inhibitor can combine with ES, and S can combine with EI to form in both with ES, and S can combine with EI to form in both instances EIS. instances EIS.

This type of inhibition is not completely reversed by high This type of inhibition is not completely reversed by high substrate concentration substrate concentration since thesince the closed sequence will closed sequence will occur regardless of the substrate concentrationoccur regardless of the substrate concentration

Since the inhibitor binding site is not identical to nor does Since the inhibitor binding site is not identical to nor does it modify the active site directly, the Kit modify the active site directly, the KMM is not altered. is not altered.

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IM

max

K]I[1]S[K

]S[VV

Noncompetitive

-I

+I

-1/Vmax

(1+[I]/KI)/Vmax

1/V

1/S

FEEDBACK FEEDBACK INHIBITIONINHIBITION

The switch: Allosteric inhibitionThe switch: Allosteric inhibition

Allosteric means Allosteric means “other site”“other site”

E

Active site

Allosteric site

© 2008 Paul Billiet ODWS

Switching offSwitching off

One site fits the One site fits the substrate like other substrate like other enzymesenzymes

The other site fits The other site fits an inhibitor an inhibitor moleculemolecule

Inhibitor fits into allosteric site

Substratecannot fit into the active site

Inhibitor molecule

© 2008 Paul Billiet ODWS

These enzymes have These enzymes have two receptor sitestwo receptor sites

HOW TO SOLVE THE EQUATIONSHOW TO SOLVE THE EQUATIONS

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1. 1. CompetitiveCompetitive inhibitorinhibitor

y =1/V; x = 1/[s]y =1/V; x = 1/[s] a = 1/a = 1/VmaxVmax b = Kb = KMM(1+[I]/K(1+[I]/KII)/)/VmaxVmax

]S[K

]I[1K

]S[VV

IM

max

maxV1

]S[1

VK

]I[1K

V1

max

IM

2. Uncompetitive 2. Uncompetitive

y =1/V; x = 1/[s]y =1/V; x = 1/[s] a = (1+[I]/Ka = (1+[I]/KII)/Vmax)/Vmax b = Kb = KMM/Vmax /Vmax

IM

max

K]I[1]S[K

]S[VV

maxVK

]I[1

]S[1

maxVK

V1 IM

3. 3. Noncompetitive InhibitionNoncompetitive Inhibition

y =1/V; x = 1/[s]y =1/V; x = 1/[s] a = (1+[I]/Ka = (1+[I]/KII)/Vmax)/Vmax b = Kb = KMM(1+[I]/K(1+[I]/KII)/Vmax )/Vmax

IM

max

K]I[1]S[K

]S[VV

maxVK

]I[1

]S[1

maxVK

]I[1K

V1 II

M

SOALSOAL

Diketahui suatu Diketahui suatu reaksi enzimatis reaksi enzimatis tanpa dan dengan tanpa dan dengan inhibitor dengan [I] = inhibitor dengan [I] = 2,2.102,2.1044M.M.

Hitunglah KM dan Hitunglah KM dan Vmax tanpa dan Vmax tanpa dan dengan I serta Kdengan I serta KII

[S] V(-I) V(+I)

1*10-4 28 17

1.5*10-4 36 23

2.0*10-4 43 29

5*10-4 65 50

7.5*10-4 74 61

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