BI 421 Midterm III Studyguide

8/9/2019 BI 421 Midterm III Studyguide

http://slidepdf.com/reader/full/bi-421-midterm-iii-studyguide 1/41

BI 421 Midterm II Study Guide 1

Bisubstrate Reactions Follow One of Several Rate Equations

o Bisubstrate reaction – involve two substrates in which either transferreactions in which the enzyme catalyzes the transfer of a specific functionalgroup, X, from one of the substrates to the other

o Sequential Reaction Occur via Single Displacements

Reactions in which all substrates must combine with the enzymebefore a reaction can occur and products be released are known as

sequential reactions (single-displacement reactions)

(1) Ordered mechanism: compulsory order of substrateaddition to the enzyme

(2) Random mechanism: no preference for the order of

substrate addition

!n the ordered mechanism, the binding of the first substrate isapparently re"uired for the enzyme to form the binding site for the

second substrate, whereas in the #andom mechanism, both bindingsites are present on the free enzyme

o Ping Pong Reactions Occur via Double Displacements

Group-transfer reactions in which one or more products are released

before all substrates have been added are known as Ping Pong

reactions (double-displacement reactions)

$ote: !n %ing %ong reactions, the substrates & and ' do notencounter one another on the surface of the enzyme

Activation Energ and the Reaction !oordinate

o "ransition State "heor




he point of highest free energy is called the transition state of thesystem

& plot of free energy versus the reaction coordinate is called atransition state diagram or reaction coordinate diagram

o

he reactants and products are states of minimum free energy, and the

transition state corresponds of the highest point of the diagram

o

o where & and ' are the reactants, % and are the products, and X* represents the transition stateo

#$%& the free energy of the transition state less that of the reactants, is+nown as the free energ of activation

The greater the value of ΔG ! the slower the reaction rate

• his is because the larger the -*, the smaller the number ofreactant molecules that have sufficient thermal energy toachieve the transition state free energy

o

!n a multistep reaction, the step with the highest transition state freeenergy acts as a .bottlenec+/ and is therefore said to be the rate'

determining step of the reactiono

o !atalsts Reduce #$%

"atalysts act by providing a reaction pathway with a transition state

whose free energy is lower than that in the uncatalyzed reaction

he difference between the values of -* for the uncatalyzed andcatalyzed reactions, -*

cat, indicates the efficiency of the catalyst he rate enhancement (the ratio of the rates of the catalyzed and

uncatalyzed reactions) is given by e-*cat0#




o




$ote that a catalyst lowers the free energy barrier by the same amountfor both the forward and reverse reactions

• onse"uently, a catalyst e"ually accelerates the forward andreverse reactions

• !f -reaction 3, the reaction proceeds spontaneously from

reactants toward products4 if -reaction 5 3, the reverse reaction proceeds spontaneously #n enzyme cannot alter -reaction, it can only decrease -* to allow the

reaction to approach e$uilibrium (where the rates of the forward andreverse reactions are e$ual) more $uickly than it would in the absence

of a catalyst

• !atalsis !an Occur "hrough Pro(imit And Orientation Effects )!ataltic strategies*

o Pro(imit and Orientation: #eactants must come together with the properspatial relationship for a reaction to occur

678 'imolecular reaction of imidazole with p9nitrophenylacetate• hen the imidazole is covalently attached to the reactant, it is

2; times more effective than when it is free in solution• his rate enhancement results from both pro7imity and

orientation effects

'y simply binding substrates, enzymes facilitate their catalyzedreactions in ; ways:

(1) 6nzymes bring substrates into contact with their catalyticgroups and, in reactions with more than one substrate,with each other < pro%imity effects alone can only

enhance reaction rates by no more than a factor of &'

(2) 6nzymes bind their substrates in the proper orientations

for reaction =olecules react most readily if they have the

proper relative orientation %roperly orienting substrates can increase reaction

rates by a factor of up to >133 (6nzymes aligntheir substrates and catalytic groups so as tooptimize reactivity)

(?) harged groups may help stabilize the transition state ofthe reaction < electrostatic catalsis

he e7pulsion of water from the active siteenhances electrostatic catalysis

he charge distribution around the active sites ofenzymes guide polar substrates toward their binding site

(;) 6nzymes freeze out the relative translational androtational motions of their substrates and catalytic groups

his is an important aspect of catalysis because, inthe transition state, the reacting groups have littlerelative motion

This effect can promote rate enhancement of up to

& *




o 'ringing substrates and catalytic groups together in a reactive orientationorders them and therefor has a substantial entropic penalty

o he free energy re"uired to overcome this entropy loss is supplied by bindingenergy of the substrate(s) to the enzyme and contributes to the decreased-*8

o

@eep in mind that enzymes are dynamic molecules that may adopt a varietyof different conformations before and after binding their substrates Auch fle7ibility is essential in order for an enzyme to productively

interact with its substrates, transition state, and products during thecourse of a reaction•

• !ataltic +echanisms

o 6nzymes, li+e other catalysts, reduce the free energy of the transition state(-*)4 they stabilize the transition state of the catalyzed reaction

o he types of catalytic mechanisms (mechanistic strategies) that enzymesemploy have been classified as:

18 &cid9base catalysis

28 ovalent atalysis?8 =etal ion atalysis;8 %ro7imity and orientation effectsB8 %referential binding of the transition state comple7

•

o +uch can be learned about enzymatic reaction mechanisms by e%amining the

corresponding nonenzymatic reaction of model compounds•

o Acid'Base !atalsis Occurs B Proton "ransfer

$eneral acid catalsis is a process in which proton transfer from an

acid lowers the free energy of a reaction,s transition state•

• 67ample: @eto9enol tautomerization

•

(a) &n uncatalyzed +eto9enol tautomerization reaction occurs "uite slowlyas a result of the high free energy of its carbanion9li+e transition state

(b) %roton donation to the o7ygen atom reduces the carbanion character ofthe transition, accelerating the reaction

(c) # reaction may also be stimulated by general base catalsis if its rate is

increased by proton abstraction by a base




o Aome reactions may be simultaneously subCect to both general acid catalysisand general base catalysis < concerted acid'base

o catal,ed reactionso

o =any types of biochemical reactions are susceptible to acid and0or basecatalysis

o

he side chains of the amino acid residues Asp& $lu& -is& !s& "r&

.s, and Arg have p@Ds in or near the physiological pE range, which permits them to act as acid0base catalystso

The ability of enzymes to arrange several catalytic groups around

their substrates makes concerted acid-base catalysis a commonenzymatic mechanisms

• he catalytic activity of these enzymes is sensitive to pE,since the pE influences the state of protonation of side chainsat the active siteo

o !ovalent !atalsis /suall Requires A 0ucleophile

!ovalent catalsis accelerates reaction rates through the transient

formation of a catalyst-substrate covalent bond

• he covalent bond is formed by the reaction of a nucleophilicgroup on the catalyst with an electrophilic group on thesubstrate < nucleophilic catalsis

o

o he amine (nucleophile) attac+s the carbonyl group to form aSchiff base (imine bond)

o

o 67ample: Fecarbo7ylation of acetoacetateo

1) he nucleophilic reaction between the catalyst and the substrate toform a covalent bond

Group pKa

Terminalcarboxyl 3.1

!p "!particacid# 3.$

Glu "Glutamicacid# 4.3

%i! 6

Terminal amino &

'y!1(.5

Tyr1(.1

1(.



BI 421 Midterm II Study Guide )

2) he withdrawal of electrons from the reaction center by the nowelectrophilic catalyst

?) he elimination of the catalyst, a reaction that is essentially theverse of stage 1



BI 421 Midterm II Study Guide &

The nucleophilicity of a substance is closely related to its basicity

• he mechanism of nucleophilic catalysis resembles that of base catalysis e7cept that, instead of abstracting a proton fromthe substrate, the catalyst nucleophilically attac+s thesubstrate to form a covalent bond8•

&n important aspect of covalent catalysis is that the more stable thecovalent bond formed! the less easily it can decompose in the final

steps of a reaction

• & good covalent catalyst must therefore combine theseemingly contradictory properties of high nucleophilicity andthe ability to form a good leaving group to easily reverse the bond formation step

• -roups with high polarizability (highly mobile electrons),such as imidazole and thiol groups, have these propertiesma+ing them good covalent catalysts

• Gunctional groups: unprotonated amino group of Hys, the

imidazole group of Eis, the thiol group of ys, the carbo7ylgroup of &sp, and the hydro7yl group of Aer •

o +etal 1on !ofactors Act As !atalsts

$early 10? of all +nown enzymes re"uire metal ions for catalyticactivity < metalloen,mes

• =etalloenzymes contain tightly bound metal ion cofactorssuch as Ge2I, Ge?I, u2I, =n2I, or o2I

=etal ions participate in the catalytic process in ? maCor ways:(1) 'y binding to substrates to orient them properly for reaction(2) 'y mediating o7idation9reduction reactions through reversible

changes in the metal ionDs o7idation state(?) 'y electrostatically stabilizing or shielding negative charges

•

!n many metal ion9catalyzed reactions, the metal ion acts in the sameway as a proton to neutralize negative charge but is more effective because metal ions can be present in high concentrations at neutral pEand can have charges greater than I1•

& metal ionDs charge also ma+es its bound water molecules moreacidic than free E2J and therefore a source of JE9 ions even belowneutral pE

•

• 67ample: arbonic &nhydrase arbonic anhydrase catalyzes this reaction:

•

arbonic anhydrase contains an essential Kn 2I ion that the enzymeDsX9ray structure indicates lies at the bottom of a 1B9L9deep active sitecleft, where it is tetrahedrally coordinated by ? evolutionarilyinvariant Eis side chains•

he Kn2I ion polarizes a water molecule so that it ionizes to form JE9,which nucleophilically attac+s the substrate J2 to yield EJ?

9

•

•



BI 421 Midterm II Study Guide $

he proton produced in the reaction is shuttles to the enzyme surfacethrough base catalysis that is facilitated by a fourth Eis residue (EisM;)

he enzymeDs catalytic site is then regenerated by the binding ofanother E2J to the Kn2I ion

En,me& 2inetics& 1nhibition And !ontrol

• he study of enzymatic reaction rates < en,me 3inetics

• Reaction 2inetics

o !hemical 2inetics 1s Described B Rate Equations



BI 421 Midterm II Study Guide 1(




o Reaction Order 1ndicates "he 0umber Of +olecules Participating 1n An

Elementar Reaction

&t constant temperature, the rate of an elementary reaction is

proportional to the fre$uency with which the reacting molecules cometogether

he proportionality constant is +nown as a rate constant and issymbolized 3

Gor the elementary reaction & < %:

•

• where v is the velocity of the reaction•

•

o A Rate Equation 1ndicates "he Progress Of A Reaction As A Function Of

"ime & rate equation can be derived from the e"uations that describe the

instantaneous reaction velocity Girst order rate e"uation:

•




• !f a reaction is first order, a plot of lnN&O versus t will yield astraight line whose slope is –+ (the negative of the first9orderrate constant) and whose intercept on the lnN&O a7is is lnN&O3




• Jne of the hallmar+s of a first9order reaction is that the time for

half of the reactant initially present to decompose! its half-time

or half-life ! t 1/2 ! is a constant and hence independent of the

initial concentration of the reactant

o En,me 2inetics Often Follows "he +ichaelis'+enten Equation

#ll enzymes can be analyzed such that their reaction rates as well as

their overall efficiency can be $uantified

he overall reaction is composed of two elementary reactions in which

the substrate forms a comple7 with the enzyme that subse"uentlydecomposes to products, regenerating enzyme:

• En,me'substrate comple( 4 ES

• &ccording to this model, when the substrate concentration becomes high enough to entirely convert the enzyme to the 6Aform, the second step of the reaction becomes rate limiting andthe overall reaction rate becomes insensitive to further increasesin substrate concentration

3 5 and 3 '5 are the forward and reverse rate constants for formation of the6A comple7 (the first reaction), and 3 6 is the rate constant for thedecomposition of 6A to % (the second reaction)8 Eere we assume, forthe sa+e of mathematical simplicity, that the second reaction isirreversible4 that is, no P is converted bac3 to S

"he +ichaelis'+enten Equation Assumes "hat ES +aintains A

Stead State

• he =ichaelis9=enten e"uation describes the rate of theenzymatic reaction as a function of substrate concentration <the formation of product from 6A is a first9order process

• hus, the rate of formation of product can be e7pressed as the

product of the rate constant of the reaction yielding product andthe concentration of its immediately preceding intermediate

• he overall rate of production of 6A is the difference betweenthe rates of the elementary reactions leading to its appearanceand those resulting in its disappearance:







&ssumptions:)5* Assumption of Equilibrium

&ssume + 91 55 + 2, so that the first step of the reaction reachese"uilibrium:

• Eere, @s is the dissociation constant of the first step in theenzymatic reaction•

)6* Assumption of Stead State

& common condition that substrate is in great e7cess overenzyme (NAO 55 N6O)

N6AO remains appro7imately constant until the substrate isnearly e7hausted

Eence, the rate of synthesis of 6A must e"ual its rate ofconsumption over most of the course of the reaction

!n other words, 6A maintains a stead state and N6AO can betreated as having a constant value:

Stead state assumption

"otal En,me concentration7

"he +ichaelis constant& 2 +

• Aolve for N6AO




•



BI 421 Midterm II Study Guide 1)

• 1nitial velocit (89):

•

• !n order to meet the conditions of the steady state assumption,

the concentration of the substrate must be much greater than theconcentration of the enzyme, which allows each enzymemolecule to repeatedly bind a molecule of substrate and convertit to product, so that N6AO is constant

• +a(imal velocit of a reaction, 8ma(, occurs at high substrateconcentrations when the enzyme is saturated, that is, when it isentirely in the 6A form:

•

• ombining initial velocity and ma7imal velocity e"uations:

•

o This e%pression! the Michaelis-Menten equation ! is the

basic e$uation of enzyme kinetics .t describes a

rectangular hyperbola/

•

"he +ichaelis !onstant -as A Simple Operational Definition

&t the substrate concentration at which NAO P @ =, v3 P Qma702 sothat 0 + is the substrate concentration at which the reaction

velocity is half-ma%imal

herefore, if an enzyme has a small value of @ =, it achievesma7imal catalytic efficiency at low substrate concentrations

he magnitude of @ = varies widely with the identity of theenzyme and the nature of the substrate

!t is also a function of temperature and pE

Aince @ A is the dissociation constant of the =ichaelis comple7,as @ A decreases, the enzymeDs affinity for substrate increases8@ = is therefore also a measure of the affinity of the enzyme for



BI 421 Midterm II Study Guide 1&

its substrate, provided + 20+ 1 is small compared to @ A, that is,+ 2@ 91 so that the 6A < % reaction proceeds more slowly than6A reverts to 6 I A



BI 421 Midterm II Study Guide 1$

3 cat:2 + is a +easure of !ataltic Efficienc

e can define the cataltic constant, 3 cat, of an enzyme as:

his "uantity is +nown as the turnover number of an enzyme because it is the number of reaction processes (turnovers) thateach active site catalyzes per unit time

Gor =ichaelis9=enten model reaction 6IA * 6A < 6I%o + cat P @ =

$ote that whereas + cat is a constant, Qma7 depends on theconcentration of the enzyme present in the e7periment system8 Qma7

increases as N6O increases hen NAO @ =, very little 6A is formed8 onse"uently, N6O P N6O,

so the initial velocity e"uation reduces to a second9order ratee"uation:

Eere, + cat0@ = is the apparent second9order rate constant of theenzymatic reaction4 the rate of the reaction varies directly withhow often enzyme and substrate encounter one another in solution8

Thus! k cat 10 + is therefore a measure of an enzyme,s catalytic efficiency

here is an upper limit to the value of + cat0@ =: !t can be nogreater than + 14 that is, the decomposition of 6A to 6I% can occur no more fre"uently than 6 and A come together to form 6A

o 2inetic Data !an Provide 8alues Of 8ma( And 2 +

&t very high values of NAO, the initial velocity, v3, asymptoticallyapproaches Qma7

& method for determining the values of Qma7 and @ =, which wasformulated by Eans Hineweaver and Fean 'ur+, uses the reciprocal ofthe =ichaelis9=enten e"uation:

his is a linear e"uation in 10v3 and 10NAO8 !f these "uantities are plottedto obtain the so9called .ineweaver'Bur3 or double'reciprocal plot,the slope of the line is @ =0Qma7, the 10v3 intercept is 10Qma7 and thee7trapolated 10NAO intercept is 910@ =







=oreover, for small values of NAO, small errors in v 3 lead to large errorsin 10v3 and hence to large errors in @ = and Qma7•

o Stead State 2inetics !annot /nambiguousl Establish A Reaction

+echanism

&lthough steady state +inetics provides valuable information about the

rates of buildup and brea+down of 6A, it provides little insight as to thenature of 6A

Rnfortunately, steady state +inetic measurements are incapable ofrevealing the number of intermediates in an enzyme9catalyzed reaction8

Ateady state +inetic measurements can provide a phenomenologicaldescription of enzymatic behavior, but the nature of the intermediatesremains indeterminate8 he e7istence of intermediates must be verifiedindependently through spectroscopic techni"ues

The steady state kinetic analysis of a reaction cannot unambiguously

establish its mechanism

• his is because no matter how simple, elegant, or rational a

postulated mechanism, there are an infinite number ofalternative mechanisms that can also account for the +inetic data

• Rsually it is the simpler mechanism that turns out to be correct, but this is not always the case8 Eowever, if kinetic data are not

compatible with a given mechanism! then that mechanism must

be re2ected

•

• En,me 1nhibition

o =any substances alter the activity of an enzyme by combining with it in a waythat influences the binding of substrate and0or its turnover8

o 3ubstances that reduce an enzyme,s activity in this way are known as inhibitor

o !rreversible enzyme inhibitor, or inactivators , bind to the enzyme so tightlythat they permanently bloc+ the enzymeDs activity

o #eversible enzyme inhibitors diminish an enzymeDs activity by interactingreversibly with it

Aome enzyme inhibitor are substances that structurally resemble theirenzymeDs substrates, but either do not react or react very slowly8

hese substances are commonly used to probe the chemical andconformational nature of an enzymeDs active site in an effort toelucidate the enzymeDs catalytic mechanism

Jther inhibitors affect catalytic activity without interfering with thesubstrate binding

•

o !ompetitive 1nhibition 1nvolves 1nhibitor Binding At An En,me;s

Substrate Binding Site

& substance that competes directly with a normal substrate for anenzymeDs substrate9binding site is +nown as a competitive inhibitor8

• Auch an inhibitor usually resembles the substrate so that itspecifically binds to the active site but differs from the substrateso that it cannot react as the substrate does8

Product inhibition S a product of the reaction, which necessarily isable to bind to the enzymeDs active site, may accumulate and competewith substrate for binding to the enzyme in subse"uent catalytic cycles

1nhibition constant – @ !, the dissociation constant for enzyme9inhibitor binding




"ransition state analogs are particularly effective inhibitors S this is because effective catalysis often depends on an enzymeDs ability to bindto and stabilize its reactionDs transition state

& compound that mimics a transition state e7ploits these binding interactions in ways that a substrate analog cannot

Aeveral of the drugs used to bloc+ E!Q protease, an essential enzymefor production of the human immunodeficiency virus, are compoundsthat were designed to mimic the enzymeDs transition state and bind tothe enzyme with high affinity•

o "he Degree Of !ompetitive 1nhibition 8aries <ith "he Fraction Of

En,me "hat -as Bound 1nhibitor

he general model for competitive inhibition is given by the followingreaction scheme:

Eere, ! is the inhibitor, 6! is the catalytically inactive enzyme9inhibitorcomple7, and it is assumed that the inhibitor binds reversibly to theenzyme and is in rapid e"uilibrium with it so that:

# competitive inhibitor therefore reduces the concentration of free

enzyme available for substrate binding

he =ichaelis9=enten e"uation for a competitively inhibited reaction is:

• , where $ote that T, a function of the inhibitorDs concentration and itsaffinity for the enzyme, cannot be less than 1

he presence of ! ma+es NAO appear to be less than it really is (ma+es@ = appear to be larger than it really is), a conse"uence of the binding of ! and A to 6 being mutually e7clusive

Eowever, increasing =S> can overwhelm a competitive inhibitor

o !n fact, T is the factor by which 435 must be increased in

order to overcome the effect of the presence of inhibitor

o &s NAO approaches infinity, v3 approaches Qma7 for anyvalue of T

o hus, the inhibitor does not affect the enzymeDs turnover number




•




@! can be measure in the double9reciprocal form:

he double9reciprocal plots for a competitive inhibitor at variousconcentrations of ! intersect at 10Qma7 on the 10v3 a7is, a property that isdiagnostic of competitive inhibition

omparing the @ ! values of competitive inhibitors with differentstructures can provide information about the binding properties of anenzymeDs active site and hence its catalytic mechanism

o /ncompetitive 1nhibition 1nvolves 1nhibitor Binding "o "he En,me'

Substrate !omple(

!n uncompetitive inhibition, the inhibitor binds directly to the enzymesubstrate comple7, but not to the free enzyme:

he inhibitor binding site has the dissociation constant:

he binding of uncompetitive inhibitor, which need not resemblesubstrate, presumably distorts the active site! thereby rendering the

enzyme catalytically inactive he double reciprocal plots consists of a family of parallel lines with slope

@ =0Qma7, 10v3 intercepts of TD0Qma7, and 10NAO intercepts of –TD0@ =







$ote that in uncompetitive inhibition, both @ =appP@ =0TD andQma7

appPQma70TD are decreased, but that @ =app 0 Qma7app P@ =0Qma7

!n contrast to the case for competitive inhibition, adding substrate doesnot reverse the effect of an uncompetitive inhibitor because the bindingof substrate does not interfere with the binding of uncompetitive

inhibitor Rncompetitive inhibitions re"uires that the inhibitor affect the catalyticfunction of the enzyme but not its substrate binding

o +i(ed 1nhibition 1nvolves 1nhibitor Binding "o Both "he Free En,me

And "he En,me'Substrate !omple(

=any reversible inhibitors interact with the enzyme in a way thataffects substrate binding as well as catalytic acidity8 !n other words, botthe enzyme and the enzyme9substrate comple7 bind inhibitor, resultingin the following model:

his phenomenon is +nown as mi(ed inhibition )noncompetitive

inhibition)8 %resumably, a mi%ed inhibitor binds to enzyme sites that participate in both substrate binding and catalysis

he two dissociation constants for inhibitor binding:

&s in uncompetitive inhibition, the apparent values of @ = and Qma7 aremodulated by the presence of inhibitor

he name mi%ed inhibition arises from the fact that the denominator ofthe =ichaelis9=enten e"uation has the factor T multiplying @= as incompetitive inhibition and the factor TD multiplying NAO as inuncompetitive inhibition

• hus, @ =appP(T0TD)@ = and Qma7app P Qma7 0TD

!f the enzyme and enzyme9substrate comple7 bind ! with e"ual affinity,then TPTD and the @ =app value is unchanged from the @ = for the reactionin the absence of inhibitor

• !n this case, only Qma7 is affected – pure noncompetitive

inhibition

Fouble9reciprocal plots for mi7ed inhibition consist of lines that havethe slope T@ =0Qma7, a 10v3 intercept of TD0Qma7 and a 10NAO intercept of – T0T@=

• he lines for increasing values of N!O (representing increasingsaturation of 6 and 6A with !) intersect to the left of the 10v3 a7is



BI 421 Midterm II Study Guide 2)

• Gor pure noncompetitive inhibition, the lines intersect on the 10NAO a7is at 910@ =

• &s with uncompetitive inhibition, substrate binding does notreverse the effects of mi7ed inhibition

he +inetics of an enzyme inactivator (an irreversible inhibitor)resembles that of a pure noncompetitive inhibitor because theinactivator reduces the concentration of functional enzyme at allsubstrate concentrations

• onse"uently, Qma7 decreases and @= is unchanged• he double9reciprocal plots for irreversible inactivation

therefore resemble those for pure noncompetitive inhibition (thelines intersect on the 10NAO a7is)

• En,mes !atal,e Reactions B Preferentiall Binding "he "ransition State




o #n enzyme may bind the transition state of the reaction it catalyzes with greater

affinity than its substrates or products

o he original concept of transition state binding proposed that enzymesmechanically strained their substrates toward the transition state geometrythrough binding sites into which undistorted substrates did not properly fit

Auch strain promotes many organic reactions 678 he rate of the reaction

is ?1B times faster when # is E? rather than E because of thegreater steric repulsion between the E? groups and the reacting groups

o he strained reactant more closely resembles the transition state of the reactionthan does the corresponding unstrained reactant

o hus, as was first suggested by Hinus %auling and further amplified by #ichardolfenden and -ustav Hienhard, enzymes that preferentially bind the transition

state structure increase its concentration and therefore proportionally increasethe reaction rate

o he more tightly an enzyme binds its reactionDs transition state relative to thesubstrate, the greater is the rate of the catalyzed reaction relative to that of theuncatalyzed reaction4 that is, catalysis results from the preferential binding andtherefore stabilization of the transition state relative to the substrate

o he free energy difference (-6*) between an enzyme9substrate comple7 (6A)

and an enzyme9transition state comple7 (6A*) is less than the free energydifference (- $

*) between A and A* in an uncatalyzed reaction

o he rate enhancement of a catalyzed reaction is given by e-*cat0#

, where-*

cat is the difference in the values of -* for the uncatalyzed (- $

*) and thecatalyzed (-6

*) reactions8o hus, a rate enhancement of 13M , which re"uires that an enzyme bind its

transition state comple7 with 13M 9fold higher affinity than its substrate,corresponds to a ?;82 +U・mole 91 stabilization at 2BV, roughly the free energyof two hdrogen bonds




onse"uently, the enzymatic binding of a transition state by two hydrogen bonds that cannot form when the substrate first binds to the enzyme shouldresult in a rate enhancement of >13M based on this effect alone

o "hus& a good substrate does not necessaril bind to its en,me with high

affinit& but it does so on activation to the transition state

o

GischeDs loc+9and9+ey model for enzyme action applies more to transition state binding than to substrate binding




o Gor some enzymes, transition state stabilization ma+es only a minor contributionto rate enhancement

he enzyme promotes the reaction by instead stabilizing the so9called near attac+ conformation, a step along the reaction coordinate in which thereactants are properly oriented and in van der aals contact but have not

yet reached the transition state hese findings suggest that enzymes have evolved to use a variety ofstrategiesSincluding stabilizing the transition state or something leadingup to itSto accelerate chemical reactionso

• "ransition State Analogs Are En,me 1nhibitors

o .f an enzyme preferentially binds its transition state! then it can be e%pected

that transition state analogs ! stable molecules that geometrically and

electronically resemble the transition state! are potent inhibitors of the enzyme

o Gor e7ample, the reaction catalyzed by proline racemase is thought to occurvia a planar transition state

o

%roline racemase is inhibited by the planar analogs of proline, prrole'

6'carbo(late and 919%yrroline929carbo7ylate, both of which bind tothe enzyme with 1M39fold greater affinity than does proline

hese compounds are therefore thought to be analogs of the transitionstate in the proline racemase reaction

o The theory that enzymes bind transition states with higher affinity than

substrates has led to a rational basis for drug design based on theunderstanding of specific enzyme reaction mechanismso

• Effects of p- on En,me Activit

o =ost enzymes are active within only a narrow pE range, typically B to Wo his is a result of the effects of pE on a combination of factors

(1) he binding of substrate to enzyme(2) he ionization states of the amino acid residues involved in the

catalytic activity of the enzyme(?) he ionization of the substrate(;) he variation of protein structure (usually significant only at

e7tremes of pE)

+xample o, Tran!ition StateBindin-




o

hese curves reflect theionization of certain amino

acid residues that must bein a specific ionization statefor enzymatic activity8




o he observed p@Ds (the inflection points of the curve) often provide valuableclues to the identities of the amino acid residues essential for enzymatic activity

Gor e7ample, an observed p@ of >; suggests that an &sp or -lu residueis essential to the enzyme

Aimilarly, p@Ds of >M or >13 suggest the participation of a Eis or a Hys

residue, respectively Eowever, the p@ of a given acid9base group may vary by as much asseveral pE units from its e7pected value, depending on itsmicroenvironment

o Gurthermore, pE effects on an enzymatic rate may reflect denaturation of theenzyme rather than protonation of deprotonation of specific catalytic residues•

• 2inetics and "ransition State "heor

•

o his e"uation indicates that the rate of a reaction not only depends on the

concentrations of its reactants, but also decreases e7ponentially with -*

hus, the larger the difference between the free energy of the transition

state and that of the reactants (the free energy of activation)! that is the

less stable the transition state! the slower the reaction proceeds

•

o his e"uation indicates that as the temperature rises, so that there is increasedthermal energy available to drive the reacting comple7 over the activation barrier (-* ), the reaction speeds up•

• ?mogens Are 1nactive En,me Precursors

o

%roteolytic enzymes are usually biosynthesized as somewhat larger inactive precursors +nown as ,mogens (enzyme precursors – proen,mes)o hymotrypsinogen is then activated by trypsin9catalyzed cleavage of its

Arg5@'1le5 peptide bondo Ae"uential proenzyme activation ma+es it possible to "uic+ly generate large

"uantities of active enzymes in response to diverse physiological signalso ?mogens -ave Distorted Active Sites

Jn activation, the newly liberated $9terminal !le 1M residue movesfrom the surface of the protein to an internal position, where its freecationic amino group forms an ion pair with the invariant &sp 1W;,which is close to the catalytic triad

ithout this conformational change, the enzyme cannot properly bindits substrate or stabilize the tetrahedral intermediate because itsspecificity poc+et and the o7yanion hole are improperly formed

his provides further structural evidence favoring the role of preferential transition state binding in the catalytic mechanism of serine proteases

$evertheless, because their catalytic triads are structurally intact, thezymogens of serine proteases actually have low levels of enzymatic




activity, an observation that was made only after the above structuralcomparisons suggested that this might be the case•




• Serine Proteases

o %roteolytic enzymes +nown as serine proteases, so named because they have acommon catalytic mechanism involving a peculiarly reactive Aer residue

o he serine proteases include digestive enzymes as well as more specialized proteins that participate in development, blood coagulation (clotting),

inflammation, and numerous other processeso Active Site Residues <ere 1dentified B !hemical .abeling

hymotrypsin, trypsin, and elastaseSall these enzymes catalyze thehydrolysis of peptide (amide) bonds but with different specificities forthe side chains flan+ing the scissile (to be cleaved) peptide bond

hymotrypsin is specific for a bul+y hydrophobic residue proceedingthe scissile bond, trypsin is specific for a positively charged residue, andelastase is specific for a small neutral residue

hymotrypsinDs catalytically important groups were identified bychemical labeling studies

• & diagnostic test for the presence of the active site Ser of serine

proteases is its reaction with diisoproplphosphofluoridate)D1PF*, which irreversibly inactivate the enzyme• Jther Aer residues, including those on the same protein,

FJ $J react with F!%Go 6.78 reacts only with 3er 9' of chymotrypsin! thereby

demonstrating that this residue is the enzyme,s active site 3er

•

& second catalytically important residue -is @, was discoveredthrough affinit labeling

• !n this techni"ue, a substrate analog bearing a reactive groupspecifically binds at the enzymeDs active site, where it reacts toform a stable covalent bond with a nearby susceptible group –"ROCA0 -ORSE

• he affinity labeled group(s) can subse"uently be isolated andidentified

&ctive Aite Feterminationvia

%rotein =odification(affinity labeling)

roCan Eorses




hymotrypsin specifically binds tosl'.'phenlalanin

chloromethl3etone )"P!2* because of its resemblance to a %heresidue (one of chymotrypsinDs preferred substrate residues

•

• &ctive site9bound %@Ds chloromethyl+etone group is a strongal3lating agent , it reacts only with Eis B, therebyinactivating the enzyme

•

o

'Ra Structures Provide 1nformation About !atalsis& SubstrateSpecificit& and Evolution

!n all three protease structures, the catalytically essential -is @ and Ser

5@ residues are located in the enzymeDs substrate9binding site he X9ray structures also show that Asp 596, which is present in all

serine proteases, is buried in a nearby solve9inaccessible poc+et These three invariant residues form a hydrogen-bonded constellation

referred to as the catalytic triad

al+ylation




•






(?) he amine leaving group is released from the enzyme and replaced by water from the solvent

(;) he acyl9enzyme intermediate, which is highly susceptible tohydrolytic cleavage, adds water by the reversal of Atep 2, yielding asecond tetrahedral intermediate

(B) he reversal of Atep 1 yields the carbo7ylate product (the new 9terminal portion of the cleaved polypeptide chain), therebyregenerating the active enzyme8

• !n this process, water is the attac+ing nucleophile and Aer1WB is the leaving group







Serine Proteases Preferentiall Bind the "ransition State

(1) he conformational rearrangement that occurs with theformation of the tetrahedral intermediate causes the nowanionic carbonyl o7ygen of the scissile peptide to movedeeper into the active site so as to occupy a previouslyunoccupied position called the o(anion hole

(2) !t forms two E9bonds with the enzyme that cannot formwhen the carbonyl group is in its normal trigonalconformation

(?) he tetrahedral distortion permits the formation of anotherwise unsatisfied E9bond between the enzyme and the bac+bone $E group of the residue preceding the scissile peptide bond

•

• This preferential binding of the transition state (or the

tetrahedral intermediate) over the enzyme-substrate comple% or

the acyl-enzyme intermediate is responsible for much of thecatalytic efficiency of serine proteases•

.ow'Barrier -drogen Bonds +a Stabili,e the "ransition State

• he transition state of the chymotrypsin reaction is stabilizednot Cust through the formation of additional E9bonds in theo7yanion hole but possibly also by the formation of anunusually strong E9bond

• %roton transfers between E9bond groups (FSE99&) occur at physiologically reasonable rates only when the p@ of the protondonor is no more than 2 or ? pE units greater than that of the protonated form of the proton acceptor

• Eowever, when the p@Ds of the hydrogen bonding donor (F)

and acceptor (&) groups are nearly e"ual, the distinction between the brea+s down: The hydrogen atom becomes more or

less e$ually shared between then (6--:--#)

• Low-barrier hydrogen bonds (LBHBs ) are unusually short andstrong

• 67perimental evidence indicates that in the serine proteasecatalytic triad, the p@Ds of the protonated Eis and &sp are nearlye"ual, and the E9bond between Eis and &sp has an unusuallyshort $99J distance of 28M2L with the E atom nearly centered between the $ and J atoms

• his suggests that the enzyme uses the .Atrategy/ of converting

a wea+ E9bond in the initial enzyme9substrate comple7 to astrong E9bond in the transition state, thereby facilitating protontransfer while applying the difference in the free energy between the normal and low9barrier E9bonds to preferentially binding the transition state•




"he tetrahedral 1ntermediate -as Been Directl Observed

• 'ecause the tetrahedral intermediate resembles the transitionstate of the serine protease reaction, it is thought to be short9lived and unstable

• his acyl9enzyme comple7, which is stable at pE B83, e7hibits

the e7pected structure, with the substrateDs 9terminal !leresidue covalently lin+ed via an ester bond to Aer 1WB• !n the first view of a serine protease acyl9enzyme intermediate,

the acyl group is fully planar, with no distortion toward atetrahedral geometry

• & water molecule is located near the intermediateDs ester bond,E9bonded to Eis B, where it appears poised to nucleophilicallyattac+ the ester lin+age

• &t p- @9, Eis B is protonated and acts as a E9bond donor towater (it cannot function as a base catalyst at this pE)

• o assess the ne7t step of the reaction, the acyl9enzyme crystal

was immersed in a solution of p- 9 (recall that proteincrystals contain large solvent9filled spaces, so protons and othersmall substances can diffuse into and out of the crystallizedenzymeDs active site)

• he resulting deprotonation of Eis B at pE W83 triggered thehydrolytic reaction

• !n this way, the tetrahedral intermediate, the o7yanion hole doesnot undergo any change in its structure, but the peptide substratemoves within its binding poc+et and becomes distorted toward atetrahedral geometry

• he tetrahedral intermediate has the e7pected shape, similar to

+now transition state analog inhibitors• Eowever, it does not bind so tightly to the amide group of the

o7yanion hole that it would not be able to subse"uentlydissociate8

Documents

BI 421 Midterm III Studyguide