PHARMACEUTICALBIOCHEMISTRY

FA31112sks
TutusGusdinarKartawinata
Pharmacochemistry ResearchGroup
SCHOOLOFPHARMACY
INSTITUTTEKNOLOGIBANDUNG
gusdinar@fa.itb.ac.id
4
th
WeekLecture
TOPICS:
TheBehaviorofProteins:
Enzymes,MechanismsandControl
Controloftheactivityofindividualenzymes
Theactivityofanenzymecanberegulatedin
twobasicways:
byalterationofthekineticconditionsunder
whichtheenzymeisoperating;
byalterationoftheamountoftheactiveform
oftheenzymepresentbypromoting
enzymesynthesis,enzymedegradationorthe
chemicalmodificationoftheenzyme.
ChapterOutline
1.TheBehaviorofAllostericEnzymes
Howareallostericenzymescontrolled?
2.TheConcertedandSequentialModelsforAllostericEnzymes
Whatistheconcertedmodelforallostericbehavior?
Whatisthesequentialmodelforallostericbehavior?
3.ControlofEnzymeActivitybyPhosphorylation
Doesphosphorylationalwaysincreaseenzymeactivity?
4.Zymogens
5.TheNatureoftheActiveSite
Howdowedeterminetheessentialaminoacidresidues?
Howdoesthearchitectureoftheactivesiteaffectcatalysis?
Howdothecriticalaminoacidscatalyzethechymotrypsinreaction?
6.ChemicalReactionsInvolvedinEnzymeMechanisms
Whatarethemostcommontypesofreactions?
7.TheActiveSiteandTransitionStates
Howdowedeterminethenatureofthetransitionstate?
8.Coenzymes
1.TheBehaviorofAllostericEnzymes
Schematic of a pathways showing feedback inhibition
Organization of aspartate transcarbamoylase
showing the two catalytic trimers and the three regulatory dimers.
Summary
1.Allostericenzymesexhibitdifferentbehaviors
comparedtononallostericenzymes,andthe
MichaelisMentenequationsarenotapplicable.
2.Aplotofvelocityversus[S]foranallostericenzyme
hasasigmoidalshape.
3.Onetypeofcontroloftenseenwithallosteric
enzymesiscalledfeedbackinhibition.
4.Inhibitorsandactivatorscancontroltheactivityof
anallostericenzyme.
2.TheConcertedandSequentialModels
forAllostericEnzymes
Summary
1.Thetwoprincipalmodelsforallostericenzymebehaviorarecalledthe
concertedmodelandthesequentialmodel.
2.Intheconcertedmodel,theenzymeisthoughtofasbeinginataut
form,T,orarelaxedform,R.Allsubunitsarefoundinoneortheother,
andanequilibriumexistsbetweentheTandRforms.
3.SubstratebindsmoreeasilytotheRformthantotheTform,inhibitors
stabilizetheTform,andactivatorsstabilizetheRform.
4.Inthesequentialmodel,subunitsoftheenzymecanchangesequentially
fromtheTformtotheRformandbackagain.
5.Bindingofonemoleculeofsubstratetoonesubunitstimulatesthe
transitionofthesubunittotheRform,whichthenstimulatesanother
subunittochangetotheRform.
6.Bindingofinhibitortoonesubunitinducesachangeintheothersubunits
toaformwithloweraffinityforthesubstrate.Bindingofanactivatorto
onesubunitinducesashift
3.ControlofEnzymeActivityby
Phosphorylation
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.
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.
Summary
1.Manyenzymesarecontrolledbyphosphorylation.
2.Enzymescalledkinasesusehighenergymolecules,
suchasATP,totransferaphosphatetoaspecific
residueinanenzyme.
3.Theseaminoacidresiduesareusuallyserine,
threonine,ortyrosineresidues.
4.Insomecases,phosphorylationincreasesthe
activityofanenzyme,whileinothercasesit
decreasesit.
4.Zymogens
The proteolytic activation of chymotrypsinogen.
Summary
1.Zymogensareinactiveprecursorsofanenzyme.
2.Azymogenisconvertedtotheactiveformbythe
irreversiblecleavageofspecificpeptidebondsin
theprotein.
3.Manydigestiveenzymes,suchastrypsinand
chymotrypsin,areinitiallyproducedaszymogens.
Theybecomeactiveonlyafterarrivingattheirfinal
destination.
5.TheNatureoftheActiveSite
Diisopropylphosphofluoridate (DIPF) labels the active-site serine of chymotrypsin
The labeling of the active-site histidine of chymotrypsin by TPCK
Thetertiarystructureofchymotrypsinplacestheessentialaminoacidresidues
closetooneanother.Theyareshowninblueandred
Otherimportantpiecesofinformationaboutthethree
dimensionalstructureoftheactivesiteemergewhena
complexisformedbetweenchymotrypsin andasubstrate
analogue.Whenonesuchsubstrateanalog,formylL
tryptophan,isboundtotheenzyme,thetryptophanside
chainfitsintoahydrophobicpocketnearserine195.
Thistypeofbindingisnotsurprising,inviewofthespecificityof
theenzymeforaromaticaminoacidresiduesatthecleavage
site.
TheresultsofXraycrystallographyshow,inadditiontothe
bindingsiteforaromaticaminoacidsidechainsofsubstrate
molecules,adefinitearrangementoftheaminoacidside
chainsthatareresponsibleforthecatalyticactivityofthe
enzyme.Theresiduesinvolvedinthisarrangementareserine
195andhistidine57.
Themechanismofchymotrypsinaction.Inthefirststageofthereaction,thenucleophileserine
195attacksthecarbonylcarbonofthesubstrate.Inthesecondstage,wateristhenucleophile
thatattackstheacylenzymeintermediate.Notetheinvolvementofhistidine57inbothstages
ofthereaction.
Summary
1.Theuniqueorientationoftheaminoacidsinthe
activesitepromotethecatalysisofachemical
reaction.
2.Tounderstandthecatalyticmechanism,thecritical
aminoacidsintheactivesitemustbedetermined.
Labelingreagentsareoftenusedforthispurpose.
3.Histidine57andserine195playthemostimportant
rolesinthemechanismofchymotrypsinaction.
6.ChemicalReactionsInvolvedinEnzyme
Mechanisms
Conceptssuchasnucleophilicattackandacidcatalysiscommonly
enterintodiscussionsofenzymaticreactions.
Nucleophilicsubstitutionreactionsplayalargeroleinthestudyof
organicchemistry,andtheyareexcellentillustrationsofthe
importanceofkineticmeasurementsindeterminingthe
mechanismofareaction.
Anucleophile isanelectronrichatomthatattacksanelectron
deficientatom.
ZisthenucleophileandXiscalledaleavinggroup.
Inbiochemistry, thecarbonofacarbonylgroup(C=O)isoftenthe
atomattackedbythenucleophile.Commonnucleophilesarethe
oxygens ofserine,threonine,andtyrosine.
Iftherateofthereactionshownhereisfoundtodependsolelyon
theconcentrationoftheR:X,thenthenucleophilicreactionis
calledanSN1(substitutionnucleophilicunimolecular). AnSN1
reactionfollowsfirstorder kinetics.
IfthenucleophileattackstheR:XwhiletheXisstillattached,then
boththeconcentrationofR:Xandtheconcentrationof:Zwillbe
important.Thisreactionwillfollowsecondorderkineticsandis
calledanSN2reaction(substitutionnucleophilicbimolecular).
ThedifferencebetweenSN1andSN2isveryimportantto
biochemistsbecauseitexplainsmuchaboutthe
stereospecificity oftheproductsformed.
AnSN1reactionoftenleadstolossofstereospecificity.Because
theleavinggroupisgonebeforetheattackinggroupenters,
theattackinggroupcanoftenendupinoneoftwo
orientations,althoughthespecificityoftheactivesitecan
alsolimitthis.
WithanSN2reaction,thefactthattheleavinggroupisstill
attachedforcesthenucleophiletoattackfromaparticular
sideofthebond,leadingtoonlyonepossiblestereospecificity
intheproduct.Thechymotrypsinnucleophilicattackswere
examplesofSN2reactions,althoughnostereochemistryis
notedbecausethecarbonylthatwasattackedbecamea
carbonylgroupagainattheendofthereactionandwas,
therefore,notchiral.
Theconceptofgeneralacidbasecatalysisdepends ondonation
andacceptanceofprotonsbygroupssuchastheimidazole,
hydroxyl,carboxyl,sulfhydryl,amino,andphenolicsidechains
ofaminoacids;allthesefunctionalgroupscanactasacidsor
bases.Thedonationandacceptanceofprotonsgivesriseto
thebondbreakingandreformationthatconstitutethe
enzymaticreaction.
Iftheenzymemechanisminvolvesanaminoaciddonatinga
hydrogenion,asinthereaction:
thenthatpartofthemechanismwouldbecalledgeneralacid
catalysis.Ifanaminoacidtakesahydrogenionfromoneof
thesubstrates,suchasinthereaction:
thenthatpartiscalledgeneralbasecatalysis.
Histidine isanaminoacidthatoftentakespartinbothreactions,
becauseithasareactivehydrogenontheimidazolesidechain
thatdissociatesnearphysiologicalpH.
Inthechymotrypsin mechanism,wesawbothacidandbase
catalysisbyhistidine.
Asecondformofacidbasecatalysisreflectsanother,more
generaldefinitionofacidsandbases.IntheLewisformulation,
anacidisanelectronpairacceptor,andabaseisanelectron
pairdonor.Metalions,includingsuchbiologicallyimportant
onesasMn
2+
,Mg
2+
,andZn
2+
,areLewisacids.Thus,theycan
playaroleinmetalioncatalysis(alsocalledLewisacidbase
catalysis).
TheinvolvementofZn
2+
intheenzymaticactivityofcarboxypep
tidaseA isanexampleofthistypeofbehavior.Thisenzyme
catalyzesthehydrolysisof Cterminalpeptidebondsof
proteins.TheZn(II),whichisrequiredfortheactivityofthe
enzyme,iscomplexedtotheimidazolesidechainsofhistidines
69and196andtothecarboxylatesidechainofglutamate72.
Thezincionisalsocomplexedtothesubstrate.
The type of binding involved in the complex is similar to the binding that links iron
to 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.
Adefiniteconnectionexistsbetweentheconceptsofacidsandbasesandthe
ideaofnucleophilesandtheircomplementarysubstances,electrophiles.
ALewisacidisanelectrophile,andaLewisbaseisanucleophile.
Catalysisbyenzymes,includingtheirremarkablespecificity,is
basedonthesewellknownchemicalprinciplesoperatingina
complexenvironment.
Thenatureoftheactivesiteplaysaparticularlyimportantrolein
thespecificityofenzymes.Anenzymethatdisplaysabsolute
specificity,catalyzingthereactionofone,andonlyone,substrate
toaparticularproduct,islikelytohaveafairlyrigidactivesite
thatisbestdescribedbythelockandkeymodelofsubstrate
binding.Themanyenzymesthatdisplayrelativespecificity,
catalyzingthereactionsofstructurallyrelatedsubstratesto
relatedproducts,apparentlyhavemoreflexibilityintheiractive
sitesandarebettercharacterizedbytheinducedfitmodelof
enzymesubstratebinding;chymotrypsin isagoodexample.
Therearestereospecificenzymes
withspecificityinwhichoptical
activityplaysarole.Thebinding
siteitselfmustbeasymmetricin
thissituation.
Iftheenzymeistobindspecificallyto
anopticallyactivesubstrate,the
bindingsitemusthavetheshape
ofthesubstrateandnotitsmirror
image.Thereareevenenzymes
thatintroduceacenterofoptical
activityintotheproduct.The
substrateitselfisnotoptically
activeinthiscase.Thereisonly
oneproduct,whichisoneoftwo
possibleisomers,notamixtureof
opticalisomers.
Summary
1.Enzymesareknowntocatalyzefamiliarorganic
chemicalreactions.
2.Oneofthemostcommonisanucleophilic
substitutionreaction,ofwhichtherearetwo
principaltypesSN1andSN2.
3.Othercommonreactionsaregeneralacidbase
catalysisandmetalioncatalysis.
7.TheActiveSiteandTransitionStates
Thetruenatureofthetransitionstateisachemicalspecies
thatisintermediateinstructurebetweenthesubstrateand
theproduct.
Thistransitionstateoftenhasaverydifferentshapefrom
eitherthesubstrateortheproduct.Inthecaseof
chymotrypsin,thesubstratehasthecarbonylgroupthatis
attackedbythereactiveserine.
Thecarbonofthecarbonylgrouphasthreebonds,andthe
orientationisplanar.Aftertheserineperformsthe
nucleophilicattack,thecarbonhasfourbondsanda
tetrahedralarrangement.
Thistetrahedralshapeisthetransitionstateofthereaction,
andtheactivesitemustmakethischangemorelikely.
Howdowedeterminethenatureofthetransition
state?
Thefactthattheenzymestabilizesthetransitionstate
hasbeenshownmanytimesbytheuseoftransition
stateanalogs,whicharemoleculeswithashapethat
mimicsthetransitionstateofthesubstrate.
ProlineracemasecatalyzesareactionthatconvertsL
prolinetoDproline.Intheprogressofthereaction,
thecarbonmustchangefromatetrahedral
arrangementtoaplanarform,andthenbackto
tetrahedral,butwiththeorientationoftwobonds
reversed.
Aninhibitorofthereactionispyrrole2
carboxylate,achemicalthatisstructurally
similartowhatprolinewouldlooklikeatits
transitionstatebecauseitisalwaysplanaratthe
equivalentcarbon.Thisinhibitorbindstoproline
racemase160timesmorestronglythanproline
does.Transitionstateanalogshavebeenused
withmanyenzymestohelpverifyasuspected
mechanismandstructureofthetransitionstate
aswellastoinhibitanenzymeselectively.
TheAbzymes
In1969,WilliamJencksproposedthatanimmunogen(amoleculethatelicitsan
antibodyresponse)wouldelicitantibodieswithcatalyticactivityifthe
immunogenmimickedthetransitionstateofthereaction.
RichardLernerandPeterSchultz,whocreatedthefirstcatalyticantibodies,
verifiedthishypothesisin1986.Becauseanantibodyisaproteindesignedto
bindtospecificmoleculesontheimmunogen,theantibodyis,inessence,a
fakeactivesite.Forexample,thereactionofpyridoxalphosphateandanamino
acidtoformthecorrespondingketoacidandpyridoxaminephosphateisa
veryimportantreactioninaminoacidmetabolism.Themolecule,Na(5'
phosphopyridoxyl)Llysineservesasatransitionstateanalogforthisreaction.
Whenthisantigenmoleculewasusedtoelicitantibodies,theseantibodies,or
abzymes,hadcatalyticactivity.Thus,inadditiontohelpingtoverifythenature
ofthetransitionstateormakinganinhibitor,transitionstateanalogsnowoffer
thepossibilityofmakingdesignerenzymestocatalyzeawidevarietyof
reactions.
8.Coenzymes
Cofactorsarenonprotein substancesthattakepartinenzymaticreactions
andareregeneratedforfurtherreaction.Metalionsfrequentlyplaysucha
role,andtheymakeuponeoftwoimportantclassesofcofactors.
Theotherimportantclass(coenzymes)isamixedbagoforganiccompounds;
manyofthemarevitaminsoraremetabolicallyrelatedtovitamins. Because
metalionsareLewisacids(electronpairacceptors),theycanactasLewis
acidbasecatalysts.Theycanalsoformcoordinationcompoundsbybehaving
asLewisacids,whilethegroupstowhichtheybindactasLewisbases.
Coordinationcompoundsareanimportantpartofthechemistryofmetal
ionsinbiologicalsystems,asshownbyZn(II)incarboxypeptidase andbyFe(II)
inhemoglobin.Thecoordinationcompoundsformedbymetalionstendto
havequitespecificgeometries,whichaidinpositioningthegroupsinvolvedin
areactionforoptimumcatalysis.
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.
Nicotinic acid is another name for the vitamin niacin. The adeninesugar
phosphate portion of the molecule is structurally related to nucleotides.
FormsofvitaminB6.ThefirstthreestructuresarevitaminB6itself,andthelasttwo
structuresshowthemodificationsthatgiverisetothemetabolicallyactivecoenzyme
TheB6vitamins(pyridoxal,pyridoxamine,andpyridoxineandtheir
phosphorylatedforms,whicharethecoenzymes)areinvolvedin
thetransferofaminogroupsfromonemoleculetoanother,an
importantstepinthebiosynthesisofaminoacids.Inthe
reaction,theaminogroupistransferredfromthedonortothe
coenzymeandthenfromthecoenzymetotheultimateacceptor.
Summary
1.Coenzymesarenonproteinsubstancesthattake
partinenzymaticreactionsandareregeneratedfor
furtherreaction.
2.Metalionscanserveascoenzymes,frequentlyby
actingasLewisacids.
3.Therearealsomanyorganiccoenzymes,suchas
NAD+andFAD,mostofwhicharevitaminsorare
structurallyrelatedtovitamins.
SummaryofChapter
Howareallostericenzymescontrolled?
Allostericenzymescanbecontrolledbymany
differentmechanisms,includinginhibitionand
activationbyreversiblybindingmolecules.
Feedbackinhibitionisacommonwayto
regulateanallostericenzymethatispartofa
complicatedpathway.
Whatistheconcertedmodelforallosteric
behavior?
Intheconcertedmodelforallostericbehavior,
thebindingofsubstrate,inhibitor,oractivatorto
onesubunitshiftstheequilibriumbetweenan
activeformoftheenzyme,whichbindssubstrate
strongly,andaninactiveform,whichdoesnot
bindsubstratestrongly.Theconformational
changetakesplaceinallsubunitsatthesame
time.
Whatisthesequentialmodelforallosteric
behavior?
Inthesequentialmodel,thebindingofsubstrate
inducestheconformationalchangeinone
subunit,andthechangeissubsequentlypassed
alongtoothersubunits.
Doesphosphorylationalwaysincreaseenzyme
activity?
Someenzymesareactivatedorinactivated
dependingonthepresenceorabsenceof
phosphategroups.Thiskindofcovalent
modificationcanbecombinedwithallosteric
interactionstoallowforahighdegreeof
controloverenzymaticpathways.
Howdowedeterminetheessentialaminoacid
residues?
Severalquestionsariseabouttheeventsthat
occurattheactivesiteofanenzymeinthe
courseofareaction.Someofthemostimportant
ofthesequestionsaddressthenatureofthe
criticalaminoacidresidues,theirspatial
arrangement,andthemechanismofthe
reaction.TheuseoflabelingreagentsandXray
crystallographyallowsustodeterminetheamino
acidsthatarelocatedintheactivesiteand
criticaltothecatalyticmechanism.
Howdoesthearchitectureoftheactivesite
affectcatalysis?
Chymotrypsinisagoodexampleofanenzymefor
whichmostofthequestionsaboutitsmechanism
ofactionhavebeenanswered.Itscriticalamino
acidresidueshavebeendeterminedtobeserine
195andhistidine57.Thecompletethree
dimensionalstructureofchymotrypsin,including
thearchitectureoftheactivesite,hasbeen
determinedbyXraycrystallography.
Howdothecriticalaminoacidscatalyzethe
chymotrypsinreaction?
Nucleophilicattackbyserineisthemainfeature
ofthemechanism,withhistidinehydrogen
bondedtoserineinthecourseofthereaction.
Thereactiontakesplaceintwophases.Inthe
firstphase,serineisthenucleophile,andthereis
anacylenzymeintermediate.Inthesecond
phase,wateractsasthenucleophileandthe
acylenzymeintermediateishydrolyzed.
Whatarethemostcommontypesof
reactions?
Commonorganicreactionmechanisms,such
asnucleophilicsubstitutionandgeneralacid
basecatalysis,areknowntoplayrolesin
enzymaticcatalysis.
Howdowedeterminethenatureofthe
transitionstate?
Thenatureofcatalysishasbeenaidedbythe
useoftransitionstateanalogs,moleculesthat
mimicthetransitionstate.Thecompounds
usuallybindtotheenzymebetterthanthe
naturalsubstrateandhelpverifythe
mechanism.Theycanalsobeusedtodevelop
potentinhibitorsortocreateantibodieswith
catalyticactivity,calledabzymes.
Thanks