w04 behaviour of proteins ii - enzyme mechanism and control (2)(1).pdf
TRANSCRIPT
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PHARMACEUTICALBIOCHEMISTRY
FA3111
2sks
TutusGusdinarKartawinata
Pharmacochemistry ResearchGroup
SCHOOLOF
PHARMACY
INSTITUTTEKNOLOGIBANDUNG
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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