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LECTURE 4:REACTION MECHANISM &

INHIBITORS

Chymotrypsin

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LECTURE OUTCOMES

After mastering the present lecture materials,students will be able to1. to explain reaction mechanisms of between enzyme

and substrate2. to explain the influence of irreversible inhibitors on

enzymatic reactions3. to explain the influence of reversible inhibitors on

enzymatic reactions4. to explain competitive, uncompetitive, noncompetitiive

inhibition of enzymatic reactions5. to calculate KM and Vmax of reactions catalyzed by

enzymes with the presence of inhibitors

LECTURE LAYOUT1. REACTION MECHANISMS1.1 Sequential Reactions1.2 Ping-Pong Reactions1.3 Kinetics of Bi-Bi Reactions

2. REACTON INHIBITION2.1 Irreversible Inhibition2.2 Reversible Inhibition2.2.1 Competitive inhibition,2.2.2 Uncompetitive inhibition.2.2.3 Noncompetitive inhibition2.3 Feedback Inhibition

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1. REACTION MECHANISMS Most biochemical reactions are not simple-,

single-substrate reactions, but typicallyinvolve two or three substrates that combineto release multiple products.

Yet, enzymatic reactions involving twosubstrates and yielding two productsaccount for ~60% of known biochemicalreactions.

Bi-Bi reactions, transferring groupscatalyzed by enzymes, fall under two majormechanistic classifications.

- Sequential reactions- Ping pong reactions

1.1 Sequential reactions Reactions in which all substrates combine

with the enzyme before a reaction can occurand products be released are known assequential reactions which is also calledsingle-displacement reactions.

Sequential reactions can be further classifiedinto 2 types :(a) Ordered Mechanism is that with a compulsory

order of substrate addition to the enzyme

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(b) Random mechanism is that with no preferencefor the order of substrate addition

Sequential Reactions: Ordered MechanismAll substrates must combine with enzyme beforereaction can occur

Bisubstrate reactions

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Sequential Reactions: Random Mechanism

1.2 Ping pong reactions Ping pong reactions are those in which one

or more products are released before allsubstrates have been added.

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1.3 Kinetics of Bi-Bi Reactions tSteady state kinetic measurements can be

utilized to distinguish among the foregoingbisubstrate mechanisms. For doing so, onemust first derive their rate equations.

This can be done in much the same way asfor single-substrate-enzymes, i.e., solving aset of simultaneous linear equationsconsisting of an equation expressing thesteady state condition for each kineticallydistinct enzyme complex plus one equationrepresenting the conservation condition forthe enzyme.

The rate equations for the above-describedbisubstrate mechanisms in the absence ofproducts are given below in doublereciprocal form.For ordered Bi-Bi reactions:

Y = a + bX1 + cX2 + dX3Y = 1/V, a = 1/Vm, b = KM

A/Vm, c =KM

B/Vm, d = KSAKM

B/Vm, X1 = 1/[A], X2 =1/[B], and X3 = 1/([A][B])

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For random Bi Bi reactions :

Y = a + bX1 + cX2 + dX3Y = 1/V, a = 1/Vm, b = KS

AKMB/(VmKS

B), c =KM

B/Vm, d = KSAKM

B/Vm, X1 = 1/[A], X2 =1/[B], and X3 = 1/([A][B])

For ping pong Bi Bi reactions :

2. REACTION INHIBITION An important number of compounds have the

ability to combine with certain enzymes in eithera reversible or irreversible manner, and thereby1. block the enzyme, but do not usually destroy it2. reduce the rate of enzymic reactions3. work specifically in general, and at low concentrations

Such compounds are called INHIBITORS andinclude;- drugs,- antibiotics,- poisons,

- anti metabolites- products of enzymic

reactions.

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30S1 32 GTP

1 2 3 GTP

Initiation Factors

mRNA

3

12 GTP

30SInitiationComplex

f-met-tRNA

Spectinomycin

Aminoglycosides

12

GDP + Pi 50S

70SInitiationComplex

AP

Inhibition ofProtein Synthesis

Two general classes of inhibitors arerecognized ;1. Irreversible2. Reversible

2.1 Irreversible An irreversible inhibitor forms a covalent bond with

a specific function, usually an amino acid residue,which may, in some manner, be associated withthe catalytic activity of the enzyme.- Many examples of enzyme inhibitors covalently bind

not at the active site, but physically block the activesite

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- The inhibitor cannot be released by dilution ordialysis; kinetically, the concentration and hence thevelocity of active enzyme is lowered in proportion tothe concentration of the inhibitor and thus the effectis that of noncompetitive inhibition.

IRREVERSIBLE INHIBITORS

Examples of irreversible inhibitors include;- DIFP (diisopropyl fluorophosphate) which reacts

irreversibly with serine proteases, chymotrypsin- Iodoacetate which reacts with essential sulfhydryl

group of an enzyme such as triose phosphatedehydrogenase:E-SH+ICH2COOH E-SCH2COOH+HI

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2.2 Reversible Inhibition As the term implies, this type of inhibition

involves equilibrium between the enzyme andthe inhibitor, the equilibrium constant (Ki) beinga measure of the affinity of the inhibitor for theenzyme.

Three distinct types of reversible inhibition areknown;- Competitive inhibition,- Uncompetitive inhibition- Noncompetitive inhibition

2.2.1 Competitive Inhibition Compounds that may or may not be structurally

related to the natural substrate combinereversibly with the enzyme at or near the activesite

The inhibitor andthe substratetherefore competefor the same siteaccording to thereaction as shownon the right side.

ES and EI complexes are formed, butEIS complexes are never produced.

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● One can conclude that high concentrations ofsubstrate will overcome the inhibition by causing thereaction sequence to swing to the right. The velocityof reaction can be calculated by the followingequation

Competitive

The rate of reaction can be calculated from thefollowing equation.

Among other enzymes that may undergo competitiveinhibition is succinic dehydrogenase, which readilyoxidizes succinic acid to fumaric acid.

If increasing concentrations of malonic acid, whichclosely resembles succinic acid in structure, areadded, however, succinic dehydrogenase activity fallsmarkedly

This inhibition cannow be reversed byincreasing in turnthe concentration ofthe substratesuccinic acid.

Competitive Inhibition

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2.2.2 Uncompetitive Inhibition Compounds that combine only with the ES

complex, not with the free enzyme, are calleduncompetitive inhibitors. The inhibition is notovercome by high substrate concentrations.

HIV protease in a complexwith the protease inhibitorritonavir

The structure of theprotease is shown by thered, blue and yellowribbons. The inhibitor isshown as the smaller ball-and-stick structure near thecentre. Created from PDB

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

Peptide-based proteaseinhibitor ritonavir

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KM value is consistently smaller than the KM valueof the uninhibited reaction which implies that S ismore effectively bound to the enzyme in thepresence of the inhibitor.

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

Uncompetitive

2.2.3 Noncompetitive Inhibition Compounds that reversibly bind with either the

enzyme or the enzyme substrate complex aredesignated as noncompetitive inhibitors

Noncompetitive inhibition therefore differs fromcompetitive inhibition in that the inhibitor cancombine with ES, and S can combine with EI to formin both instances EIS.

This type of inhibition is not completely reversed byhigh substrate concentration since the closedsequence will occur regardless of the substrateconcentration

Since the inhibitor binding site is not identical to nordoes it modify the active site directly, the KM is notaltered.

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Mix inhibition

Noncompetitive

2.3 Feedback Inhibition (Allosteric Effectors) The activity of some enzymes is controlled by

certain molecules binding to a specific regulatory(or allosteric) site on the enzyme, distinct fromthe active site.

Different molecules can either inhibit or activatethe enzyme, allowing sophisticated control of therate. Only a few enzymes can do this, and theyare often at the start of a long biochemicalpathway.

They are generally activated by the substrate ofthe pathway and inhibited by the product of thepathway, thus only turning the pathway onwhen it is needed.

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The switch: Allosteric inhibition

Allosteric means “other site”

E

Active site

Allostericsite

© 2008 Paul Billiet ODWS

Switching off These enzymes

have two receptorsites

One site fits thesubstrate like otherenzymes

The other site fitsan inhibitormolecule

Inhibitor fits intoallosteric site

Substratecannot fit intothe active site

Inhibitormolecule

© 2008 Paul Billiet ODWS

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This process is known as feedbackinhibition.

Feedback Inhibition

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HOW TO SOLVE THE EQUATIONS

1. Competitive inhibitor

y =1/V; x = 1/[s] a = 1/Vmax b = KM(1+[I]/KI)/Vmax

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2. Uncompetitive

y =1/V; x = 1/[s] a = (1+[I]/KI)/Vmax b = KM/Vmax

3. Noncompetitive Inhibition

y =1/V; x = 1/[s] a = (1+[I]/KI)/Vmax b = KM(1+[I]/KI)/Vmax

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QUIZ (10 min)1. How is enzyme specificity achieved ?2. Calculate Vmax & KM from the following data, and does the

reaction obey Michaelis-Menten kinetics ?

[DNA]mol total

nucleotides/L

Free nucleotides in solution,V (pmol/L)

0 min 10 min

1.0 x 10-5 0.05 5.1

1.0 x 10-6 0.04 4.5

1.0 x 10-7 0.06 3.2

1.0 x 10-8 0.04 1.4

1.0 x 10-9 0.04 0.23

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ANSWERS1. The enzyme specificity is achieved

through the characteristic of active site2. Vmax = 4.36695

KM = 2.2E-08R2 = 0.999864, so the reaction obeysMichaelis-Menten kinetics

SOAL

Diketahui suatureaksi enzimatistanpa dan denganinhibitor dengan [I] =2,2.104M.

Hitunglah KM danVmax tanpa dandengan I serta KI

[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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Km

Competitive Non-competitive Uncompetitive

Dire

ct P

lots

Dou

ble

Rec

ipro

cal

Vmax Vmax

Km Km’ [S], mM

vo

[S], mM

vo

I I

Km [S], mM

Vmax

I

Km’

Vmax’Vmax’

Vmax unchangedKm increased

Vmax decreasedKm unchanged Both Vmax & Km decreased

I

1/[S]1/Km

1/vo

1/ Vmax

ITwo parallellines

I

Intersectat X axis

1/vo

1/ Vmax

1/[S]1/Km 1/[S]1/Km

1/ Vmax

1/vo

Intersectat Y axis

= Km’

Juang RH (2004) BCbasics

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Example of a suicide inhibitor

9/28/05

Diisopropylfluorophosphate(DIFP) forms a covalentbond with an active -siteresidue (Ser) of the enzymechymotrypsin.

Every molecule that reactsis inactivated irreversibly.

Here, a key active site Seris bound irreversibly to theinhibitor, preventing it fromdoing its "normal" job.

12Irreversible inhibitor

The effect of enzymeinhibition

Irreversible inhibitors: Combine with thefunctional groups of the amino acids in theactive site, irreversibly

Examples: nerve gases and pesticides,containing organophosphorus, combinewith serine residues in the enzymeacetylcholine esterase

© 2008 Paul Billiet ODWS