W04 Behaviour of Proteins II - Enzyme Mechanism and Control (2)(1).pdf

8/10/2019 W04 Behaviour of Proteins II - Enzyme Mechanism and Control (2)(1).pdf

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PHARMACEUTICALBIOCHEMISTRY

FA3111

2sks

TutusGusdinarKartawinata

Pharmacochemistry ResearchGroup

SCHOOLOF

PHARMACY

INSTITUTTEKNOLOGIBANDUNG

[email protected]

4th WeekLecture


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TOPICS:

TheBehaviorofProteins:

Enzymes,Mechanisms

and

Control


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Controlof

the

activity

of

individual

enzymes

Theactivityofanenzymecanberegulatedin

twobasic

ways:

byalterationofthekineticconditionsunder

whichtheenzymeisoperating;

byalterationoftheamountoftheactiveform

oftheenzymepresentbypromoting

enzymesynthesis,

enzyme

degradation

or

the

chemicalmodificationoftheenzyme.


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ChapterOutline

1.The

Behavior

of

Allosteric

Enzymes

Howareallostericenzymescontrolled?

2.TheConcertedandSequentialModelsforAllostericEnzymes

Whatistheconcertedmodelforallostericbehavior?

Whatis

the

sequential

model

for

allosteric

behavior?

3.ControlofEnzymeActivitybyPhosphorylation

Doesphosphorylationalwaysincreaseenzymeactivity?

4.Zymogens

5.The

Nature

of

the

Active

Site

Howdowedeterminetheessentialaminoacidresidues?

Howdoesthearchitectureoftheactivesiteaffectcatalysis?

Howdothecriticalaminoacidscatalyzethechymotrypsinreaction?

6.

Chemical

Reactions

Involved

in

Enzyme

MechanismsWhatarethemostcommontypesofreactions?

7.TheActiveSiteandTransitionStates

Howdowedeterminethenatureofthetransitionstate?

8.Coenzymes


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1.TheBehaviorofAllostericEnzymes

Schematic of a pathways showing feedback inhibit ion


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Organization of aspartate transcarbamoylase

showing the two catalytic trimers and the three regulatory dimers.


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Summary

1.Allostericenzymesexhibitdifferentbehaviors

comparedto

nonallosteric

enzymes,

and

the

MichaelisMentenequationsarenotapplicable.

2.Aplotofvelocityversus[S]foranallostericenzyme

hasasigmoidal

shape.

3.Onetypeofcontroloftenseenwithallosteric

enzymesiscalledfeedbackinhibition.

4.Inhibitors

and

activators

can

control

the

activity

of

anallostericenzyme.


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2.TheConcertedandSequentialModels

forAllosteric

Enzymes


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Summary1.Thetwoprincipalmodelsforallostericenzymebehaviorarecalledthe

concertedmodelandthesequentialmodel.

2.In

the

concerted

model,

the

enzyme

is

thought

of

as

being

in

ataut

form,T,orarelaxedform,R.Allsubunitsarefoundinoneortheother,

andanequilibriumexistsbetweentheTandRforms.

3.SubstratebindsmoreeasilytotheRformthantotheTform,inhibitors

stabilizethe

Tform,

and

activators

stabilize

the

R

form.

4.Inthesequentialmodel,subunitsoftheenzymecanchangesequentially

fromtheTformtotheRformandbackagain.

5.Bindingofonemoleculeofsubstratetoonesubunitstimulatesthe

transition

of

the

subunit

to

the

R

form,

which

then

stimulates

another

subunittochangetotheRform.

6.Bindingofinhibitortoonesubunitinducesachangeintheothersubunits

toaformwithloweraffinityforthesubstrate.Bindingofanactivatorto

onesubunitinducesashift


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3.ControlofEnzymeActivityby

Phosphorylation


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Phosphorylation of the sodiumpotassium pump is involved in cycling

the membrane protein between the form that binds to sodium and the

form that binds to potassium.


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Glycogen phosphorylase activity is subject to allosteric control and covalent

modification via phosphorylation. The phosphorylated form is more active.

The enzyme that puts a phosphate group on phosphorylase is called

phosphorylase kinase.


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Summary

1.Manyenzymesarecontrolledbyphosphorylation.

2.Enzymes

called

kinases

use

high

energy

molecules,

suchasATP,totransferaphosphatetoaspecific

residueinanenzyme.

3.These

amino

acid

residues

are

usually

serine,

threonine,ortyrosineresidues.

4.Insomecases,phosphorylationincreasesthe

activityof

an

enzyme,

while

in

other

cases

it

decreasesit.


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4.Zymogens

The proteolytic activation of chymotrypsinogen.


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Summary

1.Zymogens

are

inactive

precursors

of

an

enzyme.

2.Azymogenisconvertedtotheactiveformbythe

irreversiblecleavageofspecificpeptidebondsin

theprotein.

3.Manydigestiveenzymes,suchastrypsinand

chymotrypsin,areinitiallyproducedaszymogens.

Theybecome

active

only

after

arriving

at

their

final

destination.


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5.TheNatureoftheActiveSite


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Diisopropylphosphofluoridate (DIPF) labels the active-site serine of chymotrypsin

The labeling of the active-site histid ine of chymotrypsin by TPCK


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Thetertiarystructureofchymotrypsinplacestheessentialaminoacidresidues

closeto

one

another.

They

are

shown

in

blue

and

red


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Other

important

pieces

of

information

about

the

three

dimensionalstructureoftheactivesiteemergewhena

complexisformedbetweenchymotrypsinandasubstrate

analogue.Whenonesuchsubstrateanalog,formylL

tryptophan,is

bound

to

the

enzyme,

the

tryptophan

side

chainfitsintoahydrophobicpocketnearserine195.

Thistypeofbindingisnotsurprising,inviewofthespecificityof

theenzymeforaromaticaminoacidresiduesatthecleavage

site.

TheresultsofXraycrystallographyshow,inadditiontothe

bindingsiteforaromaticaminoacidsidechainsofsubstrate

molecules,adefinite

arrangement

of

the

amino

acid

side

chainsthatareresponsibleforthecatalyticactivityofthe

enzyme.Theresiduesinvolvedinthisarrangementareserine

195andhistidine57.


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Themechanismofchymotrypsinaction.Inthefirststageofthereaction,thenucleophileserine

195attacksthecarbonylcarbonofthesubstrate.Inthesecondstage,wateristhenucleophile

thatattackstheacylenzymeintermediate.Notetheinvolvementofhistidine57inbothstages

ofthe

reaction.


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Summary

1.Theuniqueorientationoftheaminoacidsinthe

activesite

promote

the

catalysis

of

achemical

reaction.

2.Tounderstandthecatalyticmechanism,thecritical

aminoacids

in

the

active

site

must

be

determined.

Labelingreagentsareoftenusedforthispurpose.

3.Histidine57andserine195playthemostimportant

rolesin

the

mechanism

of

chymotrypsin

action.


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6.ChemicalReactionsInvolvedinEnzyme

Mechanisms


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Conceptssuchasnucleophilicattackandacidcatalysiscommonly

enterinto

discussions

of

enzymatic

reactions.

Nucleophilicsubstitutionreactionsplayalargeroleinthestudyof

organic

chemistry,

and

they

are

excellent

illustrations

of

the

importanceofkineticmeasurementsindeterminingthe

mechanismofareaction.

Anucleophileisanelectronrichatomthatattacksanelectron

deficientatom.

ZisthenucleophileandXiscalledaleavinggroup.

Inbiochemistry, thecarbonofacarbonylgroup(C=O)isoftenthe

atomattackedbythenucleophile.Commonnucleophilesarethe

oxygensofserine,threonine,andtyrosine.


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Ifthe

rate

of

the

reaction

shown

here

is

found

to

depend

solely

on

theconcentrationoftheR:X,thenthenucleophilicreactionis

calledanSN1(substitutionnucleophilicunimolecular). AnSN1

reaction

follows

first

orderkinetics.

IfthenucleophileattackstheR:XwhiletheXisstillattached,then

boththeconcentrationofR:Xandtheconcentrationof:Zwillbe

important.This

reaction

will

follow

second

order

kinetics

and

is

calledanSN2reaction(substitutionnucleophilicbimolecular).

Thedifference

between

SN1

and

SN2

is

very

important

to

biochemistsbecauseitexplainsmuchaboutthe

stereospecificityoftheproductsformed.


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AnSN1

reaction

often

leads

to

loss

of

stereospecifi

city.

Because

theleavinggroupisgonebeforetheattackinggroupenters,

theattackinggroupcanoftenendupinoneoftwo

orientations,althoughthespecificityoftheactivesitecan

alsolimit

this.

WithanSN2reaction,thefactthattheleavinggroupisstill

attachedforces

the

nucleophile

to

attack

from

aparticular

sideofthebond,leadingtoonlyonepossiblestereospecificity

intheproduct.Thechymotrypsinnucleophilicattackswere

examplesofSN2reactions,althoughnostereochemistryis

notedbecause

the

carbonyl

that

was

attacked

became

a

carbonylgroupagainattheendofthereactionandwas,

therefore,notchiral.


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Theconceptofgeneralacidbasecatalysisdepends ondonation

andacceptance

of

protons

by

groups

such

as

the

imidazole,

hydroxyl,carboxyl,sulfhydryl,amino,andphenolicsidechains

ofaminoacids;allthesefunctionalgroupscanactasacidsor

bases.Thedonationandacceptanceofprotonsgivesriseto

thebond

breaking

and

reformation

that

constitute

the

enzymaticreaction.

Iftheenzymemechanisminvolvesanaminoaciddonatinga

hydrogen

ion,

as

in

the

reaction:

thenthatpartofthemechanismwouldbecalledgeneralacid

catalysis.Ifanaminoacidtakesahydrogenionfromoneof

thesubstrates,

such

as

in

the

reaction:

thenthatpartiscalledgeneralbasecatalysis.


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Asecond

form

of

acidbase

catalysis

reflects

another,

more

generaldefinitionofacidsandbases.IntheLewisformulation,

anacidisanelectronpairacceptor,andabaseisanelectron

pairdonor.Metalions,includingsuchbiologicallyimportant

onesas

Mn2+,

Mg2+,

and

Zn2+,

are

Lewis

acids.

Thus,

they

can

playaroleinmetalioncatalysis(alsocalledLewisacidbase

catalysis).

TheinvolvementofZn2+ intheenzymaticactivityofcarboxypep

tidaseAisanexampleofthistypeofbehavior.Thisenzymecatalyzesthehydrolysisof Cterminalpeptidebondsof

proteins.TheZn(II),whichisrequiredfortheactivityofthe

enzyme,is

complexed

to

the

imidazole

side

chains

of

histidines

69and196andtothecarboxylatesidechainofglutamate72.

Thezincionisalsocomplexedtothesubstrate.


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The type of binding involved in the complex is similar to the binding that links ironto the large ring involved in the heme group. Binding the substrate to the zinc ion

polarizes the carbonyl group, making it susceptible to attack by water and allowing

the hydrolysis to proceed more rapidly than it does in the uncatalyzed reaction.


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Adefiniteconnectionexistsbetweentheconceptsofacidsandbasesandthe

ideaofnucleophilesandtheircomplementarysubstances,electrophiles.

ALewisacidisanelectrophile,andaLewisbaseisanucleophile.

Catalysisbyenzymes,includingtheirremarkablespecificity,is

based

on

these

well

known

chemical

principles

operating

in

a

complexenvironment.

Thenatureoftheactivesiteplaysaparticularlyimportantrolein

thespecificityofenzymes.Anenzymethatdisplaysabsolute

specificity,catalyzing

the

reaction

of

one,

and

only

one,

substrate

toaparticularproduct,islikelytohaveafairlyrigidactivesite

thatisbestdescribedbythelockandkeymodelofsubstrate

binding.Themanyenzymesthatdisplayrelativespecificity,

catalyzingthe

reactions

of

structurally

related

substrates

to

relatedproducts,apparentlyhavemoreflexibilityintheiractive

sitesandarebettercharacterizedbytheinducedfitmodelof

enzymesubstrate

binding;

chymotrypsinis

a

good

example.


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Thereare

stereospecific

enzymes

withspecificityinwhichoptical

activityplaysarole.Thebinding

siteitselfmustbeasymmetricin

thissituation.

Iftheenzymeistobindspecificallyto

anopticallyactivesubstrate,the

bindingsitemusthavetheshape

ofthesubstrateandnotitsmirror

image.There

are

even

enzymes

thatintroduceacenterofoptical

activityintotheproduct.The

substrateitselfisnotoptically

activeinthiscase.Thereisonly

oneproduct,whichisoneoftwo

possibleisomers,notamixtureof

opticalisomers.


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Summary

1.Enzymesareknowntocatalyzefamiliarorganic

chemicalreactions.

2.Oneofthemostcommonisanucleophilic

substitutionreaction,ofwhichtherearetwo

principaltypesSN1andSN2.

3.Othercommonreactionsaregeneralacidbase

catalysisandmetalioncatalysis.


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7.TheActiveSiteandTransitionStates

Thetruenatureofthetransitionstateisachemicalspecies

thatisintermediateinstructurebetweenthesubstrateand

the

product.

Thistransitionstateoftenhasaverydifferentshapefrom

eitherthesubstrateortheproduct.Inthecaseof

chymotrypsin,thesubstratehasthecarbonylgroupthatis

attackedby

the

reactive

serine.

Thecarbonofthecarbonylgrouphasthreebonds,andthe

orientationisplanar.Aftertheserineperformsthe

nucleophilicattack,thecarbonhasfourbondsanda

tetrahedralarrangement.

Thistetrahedralshapeisthetransitionstateofthereaction,

andtheactivesitemustmakethischangemorelikely.


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Howdowedeterminethenatureofthetransition

state?

The

fact

that

the

enzyme

stabilizes

the

transition

state

hasbeenshownmanytimesbytheuseoftransition

stateanalogs,whicharemoleculeswithashapethat

mimicsthetransitionstateofthesubstrate.

ProlineracemasecatalyzesareactionthatconvertsL

prolinetoDproline.Intheprogressofthereaction,

thecarbonmustchangefromatetrahedral

arrangementto

aplanar

form,

and

then

back

to

tetrahedral,butwiththeorientationoftwobonds

reversed.


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Aninhibitor

of

the

reaction

is

pyrrole

2

carboxylate,achemicalthatis structurally

similartowhatprolinewouldlooklikeatits

transitionstate

because

it

is

always

planar

at

the

equivalentcarbon.Thisinhibitorbindstoproline

racemase160timesmorestronglythanproline

does.Transition

state

analogs

have

been

used

withmanyenzymestohelpverifyasuspected

mechanism

and

structure

of

the

transition

state

aswellastoinhibitanenzymeselectively.


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TheAbzymes

In1969,WilliamJencksproposedthatanimmunogen(amoleculethatelicitsan

antibodyresponse)wouldelicitantibodieswithcatalyticactivityifthe

immunogenmimickedthetransitionstateofthereaction.

RichardLernerandPeterSchultz,whocreatedthefirstcatalyticantibodies,

verifiedthishypothesisin1986.Becauseanantibodyisaproteindesignedto

bindtospecificmoleculesontheimmunogen,theantibodyis,inessence,a

fakeactivesite.Forexample,thereactionofpyridoxalphosphateandanamino

acidto

form

the

corresponding

keto

acid

and

pyridoxamine

phosphate

is

a

veryimportantreactioninaminoacidmetabolism.Themolecule,Na(5'

phosphopyridoxyl)Llysineservesasatransitionstateanalogforthisreaction.

Whenthisantigenmoleculewasusedtoelicitantibodies,theseantibodies,or

abzymes,had

catalytic

activity.

Thus,

in

addition

to

helping

to

verify

the

nature

ofthetransitionstateormakinganinhibitor,transitionstateanalogsnowoffer

thepossibilityofmakingdesignerenzymestocatalyzeawidevarietyof

reactions.


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8.

CoenzymesCofactorsarenonprotein substancesthattakepartinenzymaticreactions

andareregeneratedforfurtherreaction.Metalionsfrequentlyplaysucha

role,andtheymakeuponeoftwoimportantclassesofcofactors.

Theotherimportantclass(coenzymes)isamixedbagoforganiccompounds;

manyofthemarevitaminsoraremetabolicallyrelatedtovitamins. Because

metalionsareLewisacids(electronpairacceptors),theycanactasLewis

acidbasecatalysts.

They

can

also

form

coordination

compounds

by

behaving

asLewisacids,whilethegroupstowhichtheybindactasLewisbases.

Coordinationcompoundsareanimportantpartofthechemistryofmetal

ionsinbiologicalsystems,asshownbyZn(II)incarboxypeptidase andbyFe(II)

inhemoglobin.Thecoordinationcompoundsformedbymetalionstendto

havequitespecificgeometries,whichaidinpositioningthegroupsinvolvedin

areactionforoptimumcatalysis.


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Nicotinamide adenine dinucleotide

(NAD+) is a coenzyme in many

oxidationreduction reactions. Its

structure has three partsa

nicotinamide ring, an adenine ring,

and two sugarphosphate groups

linked together. The nicotinamide ring

contains the site at which oxidation

and reduction reactions occur.


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Nicotinic acid is another name for the vitamin niacin. The adeninesugarphosphate portion of the molecule is structurally related to nucleotides.


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FormsofvitaminB6.ThefirstthreestructuresarevitaminB6itself,andthelasttwo

structuresshowthemodificationsthatgiverisetothemetabolicallyactivecoenzyme


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TheB6vitamins(pyridoxal,pyridoxamine,andpyridoxineandtheir

phosphorylatedforms,whicharethecoenzymes)areinvolvedin

thetransferofaminogroupsfromonemoleculetoanother,an

importantstepinthebiosynthesisofaminoacids.Inthe

reaction,the

amino

group

is

transferred

from

the

donor

to

the

coenzymeandthenfromthecoenzymetotheultimateacceptor.


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Summary

1.Coenzymes

are

nonprotein

substances

that

take

partinenzymaticreactionsandareregeneratedfor

furtherreaction.

2.Metal

ions

can

serve

as

coenzymes,

frequently

by

actingasLewisacids.

3.Therearealsomanyorganiccoenzymes,suchas

NAD+and

FAD,

most

of

which

are

vitamins

or

are

structurallyrelatedtovitamins.


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SummaryofChapter


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Howareallostericenzymescontrolled?

Allostericenzymescanbecontrolledbymany

differentmechanisms,includinginhibitionand

activationby

reversibly

binding

molecules.

Feedbackinhibitionisacommonwayto

regulateanallostericenzymethatispartofa

complicatedpathway.


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Whatis

the

concerted

model

for

allosteric

behavior?

Inthe

concerted

model

for

allosteric

behavior,

thebindingofsubstrate,inhibitor,oractivatorto

onesubunitshiftstheequilibriumbetweenan

activeformoftheenzyme,whichbindssubstrate

strongly,andaninactiveform,whichdoesnot

bindsubstrate

strongly.

The

conformational

changetakesplaceinallsubunitsatthesame

time.


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Whatisthesequentialmodelforallosteric

behavior?

Inthesequentialmodel,thebindingofsubstrate

inducesthe

conformational

change

in

one

subunit,andthechangeissubsequentlypassed

alongtoothersubunits.


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Doesphosphorylation

always

increase

enzyme

activity?

Someenzymesareactivatedorinactivated

dependingonthepresenceorabsenceof

phosphategroups.

This

kind

of

covalent

modificationcanbecombinedwithallosteric

interactionstoallowforahighdegreeof

controlover

enzymatic

pathways.

Howdowedeterminetheessentialaminoacid


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residues?

Severalquestionsariseabouttheeventsthat

occurattheactivesiteofanenzymeinthe

courseofareaction.Someofthemostimportant

ofthesequestionsaddressthenatureofthe

criticalamino

acid

residues,

their

spatial

arrangement,andthemechanismofthe

reaction.TheuseoflabelingreagentsandXray

crystallographyallows

us

to

determine

the

amino

acidsthatarelocatedintheactivesiteand

criticaltothecatalyticmechanism.

How does the architecture of the active site


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Howdoesthearchitectureoftheactivesite

affectcatalysis?

Chymotrypsinis

agood

example

of

an

enzyme

for

whichmostofthequestionsaboutitsmechanism

ofactionhavebeenanswered.Itscriticalamino

acidresidues

have

been

determined

to

be

serine

195andhistidine57.Thecompletethree

dimensionalstructureofchymotrypsin,including

thearchitecture

of

the

active

site,

has

been

determinedbyXraycrystallography.

H d th iti l i id t l th


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Howdothecriticalaminoacidscatalyzethe

chymotrypsinreaction?

Nucleophilicattack

by

serine

is

the

main

feature

ofthemechanism,withhistidinehydrogen

bondedtoserineinthecourseofthereaction.

Thereaction

takes

place

in

two

phases.

In

the

firstphase,serineisthenucleophile,andthereis

anacylenzymeintermediate.Inthesecond

phase,water

acts

as

the

nucleophile

and

the

acylenzymeintermediateishydrolyzed.


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Whatarethemostcommontypesof

reactions?

Commonorganicreactionmechanisms,such

asnucleophilic

substitution

and

general

acid

basecatalysis,areknowntoplayrolesin

enzymatic

catalysis.

How do we determine the nature of the


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Howdowedeterminethenatureofthe

transitionstate?

The

nature

of

catalysis

has

been

aided

by

the

useoftransitionstateanalogs,moleculesthat

mimicthetransitionstate.Thecompounds

usuallybind

to

the

enzyme

better

than

the

naturalsubstrateandhelpverifythe

mechanism.Theycanalsobeusedtodevelop

potentinhibitors

or

to

create

antibodies

with

catalyticactivity,calledabzymes.


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W04 Behaviour of Proteins II - Enzyme Mechanism and Control (2)(1).pdf