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9/16/16
Enzymesareusedalloveryourbody!
Biochemistry
Whatisanenzyme?
• Almostallenzymesareproteinsthatactas
biologicalcatalysts.
3.1)Enzymes-I
•
Introductiontoenzymestructureand
function,andfactorsinvolvingtheir
actionsandpathways
Acatalystspeedsupchemicalreactions.Enzymes
speedupbiologicalchemicalreactions.
• Enzymesarehighlyspecifictoatypeofreaction.
• Enzymesmustmaintaintheirspecificshapeinorder
tofunction.Anyalterationintheprimary,
secondary,tertiary,orquaternaryformsofthe
enzymearedetrimental.
Prof. Dr. Klaus Heese
EnzymesasBiologicalCatalysts
CO2 +H2O--->H2CO3
• Enzymes areproteins
thatincreasetherate
ofreactionby
loweringtheenergy
ofactivation
• Theycatalyzenearly
allthechemical
DG
reactionstaking
placeinthecellsof
thebody
• Enzymeshaveunique
three-dimensional
•Increasedreactionratessometimes106 to1012
increase
shapesthatfitthe
shapesofreactants
EnzymesdonotchangeDG,justthereactionrates.
(substrates)
Enzymescatalyzebystabilizingtransitionstates
• FreeenergyGofachemical
reactioncanbeplottedovertime
• Favorablereactionshavea
positivedifference(DG)infree
energybetweenthesubstrate
andproduct
• Thefreeenergyofactivationfor
thetransitionstatelimitsthe
progressofthereaction
• Enzymesactbyreducingthefree
energyofthetransitionstate
(Enzymesalterrates,notequilibria)
Functionofenzymes
Enzymeshavemanyjobs.They:
• Breakdownnutrientsintouseablemolecules.
• Storeandreleaseenergy(ATP).
• Createlargermoleculesfromsmallerones.
• Coordinatebiologicalreactionsbetweendifferent
systemsinanorganism.
•Milderreactionconditions
•Greatreactionspecificity
Naming Enzymes
• Thenameofanenzymeidentifiesthereactingsubstance
- usuallyendsin–ase
• Forexample,sucrase catalyzesthehydrolysisofsucrose
• Thenamealsodescribesthefunctionoftheenzyme
• Forexample,oxidases catalyzeoxidationreactions
• Sometimescommonnamesareused,particularlyforthe
digestionenzymessuchaspepsin andtrypsin
• Somenamesdescribeboththesubstrateandthefunction
• Forexample,alcoholdehydrogenase oxidesethanol
ClassificationofEnzymes
• Enzymesareclassifiedaccordingtothetypeofreactionthey
catalyze:
Class
Reactionscatalyzed
§ Oxidoreductases
Oxidation-reduction
§ Transferases
Transfergroupsofatoms
§ Hydrolases Hydrolysis
§ Lyases
§ Isomerases
§ Ligases
Addatoms/removeatomsto/froma
doublebond
Rearrangeatoms
UseATPtocombinemolecules
SystematicName
• AccordingtotheInternationalunionof
Biochemistryanenzymenamehastwoparts:
- Firstpartisthenameofthesubstratesforthe
enzyme.
- Secondpartisthetypeofreactioncatalyzedby
theenzyme.Thispartendswiththesuffix“ase”.
Example:Lactatedehydrogenase
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ECnumber
ECnumbers
Enzymesareclassifiedintosixdifferentgroups
accordingtothereactionbeingcatalyzed.The
nomenclaturewasdeterminedbytheEnzyme
Commissionin1961(withthelatestupdate
havingoccurredin1992),henceallenzymes
areassignedan“EC” number.The
classificationdoesnottakeintoaccount
aminoacidsequence(ie,homology),protein
structure,orchemicalmechanism.
• ECnumbersarefourdigits,forexamplea.b.c.d,
where“a” istheclass,“b” isthesubclass,“c” is
thesub-subclass,and“d” isthesub-sub-subclass.
The“b” and“c” digitsdescribethereaction,
whilethe“d” digitisusedtodistinguishbetween
differentenzymesofthesamefunctionbasedon
theactualsubstrateinthereaction.
EC1.Oxidoreductases
• EC1.Oxidoreductases:catalyzethetransferof
hydrogenoroxygenatomsorelectronsfrom
onesubstratetoanother,alsocalledoxidases,
dehydrogenases,orreductases.Notethat
sincetheseare‘redox’ reactions,anelectron
donor/acceptorisalsorequiredtocomplete
thereaction.
EC4.Lyases
• EC4.Lyases – catalyzenon-hydrolytic(coveredin
EC3)removaloffunctionalgroupsfrom
substrates,oftencreatingadoublebondinthe
product;orthereversereaction,i.e. additionof
functiongroupsacrossadoublebond.
• A-B→A=B+X-Y
• Example:forAlcohol:NAD+oxidoreductase:EC
numberis1.1.1.1
EC2.Transferases
• EC2.Transferases – catalyzegrouptransfer
reactions,excludingoxidoreductases (which
transferhydrogenoroxygenandareEC1).
Theseareofthegeneralform:
• A-X+B↔BX+A
EC5.Isomerases
• EC5.Isomerases – catalyzesisomerization
reactions,includingracemizations andcistransisomerizations.
ClassificationofEnzymes
TheSixClasses
Enzymesareclassifiedaccordingtothetypeofreactiontheycatalyze:
•
•
•
•
•
•
EC1.Oxidoreductases
EC2.Transferases
EC3.Hydrolases
EC4.Lyases
EC5.Isomerases
EC6.Ligases
Additionalinformationonthesubclasses,thesub-subclassesandsub-subsubclasses(ie,fullenzymeclassificationandnames)canbefoundatthe
referencedweblink.
• FromtheWebversion,
http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html
EC3.Hydrolases
• EC3.Hydrolases– catalyzehydrolytic
reactions.Includeslipases,esterases,
nitrilases,peptidases/proteases.Theseareof
thegeneralform:
• A-X+H2 O↔X-OH+HA
EC6.Ligases
• EC6.Ligases-- catalyzesthesynthesisof
various(mostlyC-X)bonds,coupledwiththe
breakdownofenergy-containingsubstrates,
usuallyATP
XY
• Includesdecarboxylasesandaldolases inthe
removaldirection,andsynthasesintheaddition
direction.
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Oxidoreductases,Transferases andHydrolases
Lyases,Isomerases andLigases
Enzymeactionoverview
• Enzymesarelargemoleculesthathaveasmallsection
dedicatedtoaspecificreaction.Thissectioniscalled
theactive site.
• Theactivesitereactswiththedesiredsubstance,called
thesubstrate.
• Thesubstratemayneedanenvironmentdifferentfrom
themostlyneutralenvironmentofthecellinorderto
react.Thus,theactivesitecanbemoreacidicorbasic,
orprovideopportunitiesfordifferenttypesofbonding
tooccur,dependingonwhattypeofsidechainsare
presentontheaminoacidsoftheactivesite.
ActiveSiteofanEnzyme
• Theactivesite isaregion
withinanenzymethatfits
theshapeofsubstrate
molecules
• Aminoacidside-chains
aligntobindthesubstrate
throughH-bonding,saltbridges,hydrophobic
interactions,etc.
• Productsarereleased
whenthereactionis
complete(theynolonger
fitwellintheactivesite)
ExampleofanEnzyme-CatalyzedReaction
• Thereactionforthesucrase catalyzedhydrolysisofsucroseto
glucoseandfructosecanbewrittenasfollows:
E +S D ES ® E +P1 +P2
whereE =sucrase,S =sucrose,P1 =glucoseandP2 =fructose
EnzymeSpecificity
Enzyme-CatalyzedReactions
• Enzymeshavevaryingdegreesofspecificity forsubstrates
• Enzymesmayrecognizeandcatalyze:
- asinglesubstrate
- agroupofsimilarsubstrates
- aparticulartypeofbond
• Whenasubstrate(S)fitsproperlyinanactivesite,an
enzyme-substrate(ES)complex isformed:
E +S D ES
• WithintheactivesiteoftheES complex,thereaction
occurstoconvertsubstratetoproduct(P):
ES ® E +P
• Theproductsarethenreleased,allowinganother
substratemoleculetobindtheenzyme
- thiscyclecanberepeatedmillions(orevenmore)times
perminute
• Theoverallreactionfortheconversionofsubstrateto
productcanbewrittenasfollows:
E +S D ES ® E +P
Isoenzymes
• Isoenzymes aredifferentformsofanenzymethatcatalyze
thesamereactionindifferenttissuesinthebody
- theyhaveslightvariationsintheaminoacidsequences
ofthesubunitsoftheirquaternarystructure
• Forexample,lactatedehydrogenase(LDH),whichconverts
lactatetopyruvate,consistsoffiveisoenzymes
DiagnosticEnzymes
• Thelevelsofdiagnosticenzymes inthebloodcanbeused
todeterminetheamountofdamageinspecifictissues
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Factorsthataffectenzymeaction
DenaturingEnzymes
(=conformationchange=changeofproteins’3Dstructure)
• Whenanenzymeisdenatureditisdamaged.
• Denaturingchangestheshape.
• Withoutthecorrectshapeenzymeswon’t
functionproperly.
• HOWareenzymesdenatured?
– Temperature
– pH
pHandEnzymeActivity
• Enzymes aremostactiveatoptimumpH
• Aminoacidswithacidicorbasicside-chainshavethe
properchargeswhenthepHisoptimum
• ActivityislostatloworhighpHastertiarystructureis
disrupted
EnzymeConcentrationandReactionRate
• Therateofreactionincreasesasenzymeconcentration
increases(atconstantsubstrateconcentration)
• Athigherenzymeconcentrations,moreenzymesare
availabletocatalyzethereaction(morereactionsatonce)
• Thereisalinearrelationshipbetweenreactionrateand
enzymeconcentration(atconstantsubstrateconcentration)
Factorsthataffectenzymeaction
Enzymesaremostlyaffectedbychangesintemperature
andpH.
TemperatureandEnzymeActivity
• Enzymes aremostactiveatanoptimumtemperature(usually
37°Cinhumans)
• Theyshowlittleactivityatlowtemperatures
• Activityislostathightemperaturesasdenaturationoccurs
• Toohighofatemperaturewilldenaturetheprotein
components,renderingtheenzymeuseless.
• pHrangesoutsideoftheoptimalrangewillprotonate
ordeprotonate thesidechainsoftheaminoacids
involvedintheenzyme’sfunctionwhichmaymake
themincapableofcatalyzingareaction.
OptimumpHforSelectedEnzymes
• MostenzymesofthebodyhaveanoptimumpHofabout7.4
• However,incertainorgans,enzymesoperateatlowerand
higheroptimumpHvalues
SubstrateConcentrationandReactionRate
• Therateofreactionincreasesassubstrateconcentration
increases(atconstantenzymeconcentration)
• Maximumactivity occurswhentheenzymeissaturated
(whenallenzymesarebindingsubstrate)
• Therelationshipbetweenreactionrateandsubstrate
concentrationisexponential,andasymptotes(levelsoff)
whentheenzymeissaturated
Factorsthataffectenzymeaction
Enzymesarealsoaffectedbytheconcentrationofsubstrate,
cofactorsandinhibitors,aswellasallostericregulationand
feedbackinhibition.
•
Theconcentrationof substratewilldictatehowmanyenzymes
canreact.Toomuchsubstratewillslowtheprocessuntilmore
enzymecanbemade.
•
Theavailabilityofcofactors alsodictateenzymeaction.Toolittle
cofactorswillslowenzymeactionuntilmorecofactorsareadded.
•
Aninfluxofcompetitiveornon-competitiveinhibitorswillnot
necessarilyslowtheenzymeprocess,butwillslowtheamountof
desiredproduct.
Enzymeactiontheories
• LockandKey: Thistheory,postulatedby
EmilFischerin1894,proposedthatan
enzymeis“structurallycomplementary
totheirsubstrates” andthusfittogether
perfectlylikealockandkey.Thistheory
formedthebasisofmostoftheideasof
howenzymeswork,butisnotcompletely
correct.
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Enzymeactiontheories
Lock-and-KeyModel
• Inthelock-and-keymodel ofenzymeaction:
- theactivesitehasarigidshape
- onlysubstrateswiththematchingshapecanfit
- thesubstrateisakeythatfitsthelockoftheactivesite
• Thisisanoldermodel,however,anddoesnotworkforall
enzymes
InducedFitModel
• Enzymescanformtotheshapeofits
substrate.
Enzymeactiontheories
Enzymeactiontheories
•
InducedFit: Anenzymethatisperfectlycomplementarytoits
substratewouldactuallynotmakeagoodenzymebecausethe
reactionhasnoroomtoproceedtothetransitionstateofthe
reaction. Togotocompletion,areactionmustgothroughthe
transitionstate.Inthelockandkeytheory,thesubstrateorthe
enzymecannotchangeconformationstothetransitionstate.
Therefore,enzymesmustactuallybecomplementarytothe
transitionstate sothereactionmayproceed.Thisideawas
researchedbyHaldanein1930,andLinusPaulingin1946.This
idealedtheInducedFittheory,postulatedbyDanielKoshland
in1958,wheretheenzymeitself canchangeconformationsto
facilitatethetransitionstateofthesubstrate.Thischangein
conformationoftheenzymeallowsthenecessaryfunctional
groupsattheactivesitetomoveclosertothesubstrate,
enhancingtheefficiencyofthereaction.
Enzymecofactors
• Acofactor isasubstance,thatisnotaprotein,thatmust
bindtotheenzymeinorderfortheenzymetowork.
• metalionsascofactors-- Zn2+,Fe2+,Cu2+,others
• Acofactorcanbeoforganicorigin.Anorganiccofactoris
calledacoenzyme.
InducedFitModel
• Intheinduced-fitmodel ofenzymeaction:
- theactivesiteisflexible,notrigid
- theshapesoftheenzyme,activesite,andsubstrateadjust
tomaximizethefit,whichimprovescatalysis
- thereisagreaterrangeofsubstratespecificity
• Thismodelismoreconsistentwithawiderrangeofenzymes
Enzymecofactorscont.
• Anenzymethatisbondedtoitscofactoriscalleda
holoenzyme.
• Anenzymethatrequiresacofactor,butisnotbondedto
thecofactoriscalledanapoenzyme.Apoenzymes arenot
activeuntiltheyarecomplexed withtheappropriate
cofactor.
• Cofactorsarenotpermanentlybonded.Permanently
bondedcofactorsarecalledprostheticgroups.
Coenzymes
Coenzymes:smallermoleculesthataidinenzymechemistry.
Enzymescan:
a.Carryoutacid-basereactions
b.Transientcovalentbonds
c.Charge-chargeinteractions
Enzymescannotdo:
d.Oxidation- Reduction(=Redox)reactions
e.Carbongrouptransfers
Prostheticgroup:permanentlyassociatedwithanenzyme
ortransientlyassociated.
Holoenzyme:catalyticallyactiveenzymewithcofactor.
Apoenzyme: Enzymewithoutitscofactor.
Commoncoenzymes
Manycoenzymesarederivedfromvitamins:
• NAD + (nicotinamide adeninedinucleotide); derived
fromniacin(B 3).
• CoenzymeA(CoA);derivedfrompantothenicacid
(B 5).
• FAD(flavin adeninedinucleotide);derivedfrom
riboflavin(B 2).
Commom Coenzymes
Coenzyme
Reactionmediated
Biotin
Carboxylation
Cobalamin (B12)
Alkylationtransfers
CoenzymeA
Acyltransfers
Flavin
Oxidation-Reduction
Lipoic acid
Acyltransfers
Nicotinamide
Oxidation-Reduction
Pyridoxal Phosphate
Aminogrouptransfers
Tetrahydrofolate
One-carbongrouptransfers
Thiaminepyrophosphate Aldehydetransfer
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VitaminsareCoenzymeprecursors
Thesearewatersolublevitamins.TheFatsolublevitaminsarevitaminsAand
D.
Commoncoenzymes
Humanscannotsynthesizetheseandrelayontheirpresenceinourdiets.
Thosewhohaveanunbalanceddietmaynotbereceivingasufficientsupply.
Vitamin
Coenzyme
DeficiencyDisease
Biotin
Biocytin
notobserved
Cobalamin (B12)
Cobalamin
Perniciousanemia
Folicacid
tetrahydrofolate
Neuraltubedefects
Megaloblastic anemia
Nicotinamide
Nicotinamide
Pantothenate
CoenzymeA
Pyridoxine(B6) Pyridoxal phosphate
Riboflavin(B2)
Thiamine(B1)
Niacin(niacinamide)deficiencyleadstopellagracharacterizedbydiarrhea,
dermatitisanddementia.PellagrawasendemicisSouthernUnitedStatesinthe
early20thcentury.Niacincanbesynthesizedfromtheessentialaminoacid,
tryptophan.Acorndietprevalentatthetimerestrictedtheabsorptionof
tryptophancausingadeficiency.Treatmentofcornwithbasecouldreleasethe
tryptophan(MexicanIndianstreatedcornwithCa(OH)2 beforemakingtortillas!)
•
ATP(adenosinetriphosphate);derivedfromNADHfrom
carbohydratesconsumed.(ATPcanalsoactas
neurotransmitter)
•
CTP(Cytidine triphosphate);derivedfromglutamateand
carbamoylphosphate.
•
PAPS(3'-Phosphoadenosine-5'-phosphosulfate); derivedfrom
adenosine5'-phosphosulfate(APS)andsulfateion.
Pellagra
Notobserved
Notobserved
Flavin
Thiaminepyrophosphate
Notobserved
Beriberi
Fattyacidsynthesis
Coenzymereactions
• Coenzymeshelptotransferafunctionalgrouptoamolecule.
CoenzymeAisconvertedtoacetyl-CoenzymeA
enzymeispyruvatedehydrogenase
• Forexample,coenzymeA(CoA)isconvertedtoacetyl-CoAin
themitochondriausingpyruvateandNAD+.
• Acetyl-CoAcanthenbeusedtotransferanacetyl group
(CH3CO)toaidinfattyacidsynthesis.
•
The“normal” wayanenzymefunctionsiswhenthespecific
substratebindstotheactivesiteandcreatestheproducts.
•
Asimilarsubstratecanalsobondtotheactivesitecovalently
andirreversibly.Thispreventstheenzymefromfunctioning.
Irreversibleinhibition.
•
Asimilarsubstratecanbindtotheactivesite,notpermanently,
andpreventsthedesiredsubstratefromenteringtheactivesite.
Thischangestheproductsandfunctioningoftheenzyme.Thisis
calledcompetitiveinhibition.
Amoleculecanbondtoanotherpartoftheenzymeandcausea
changeinconformation.Thischangecausestheactivesiteto
changeshapeaswell.Thischangeinshapepreventsthedesired
substratefromenteringtheactivesite.Thisiscallednoncompetitiveinhibition.
Factorsthataffectenzymeaction
• Enzymesthatcanbeactivatedwillbeaffectedbythe
amountofactivatororinhibitorattachedtoits
allostericsite.Anabundanceofanallostericactivator
willconvertmoreenzymestotheactiveformcreating
moreproduct.
• Enzymesthatarepartofametabolicpathwaymaybe
inhibitedbytheveryproducttheycreate.Thisis
calledfeedbackinhibition.Theamountofproduct
generatedwilldictatethenumberofenzymesusedor
activatedinthatspecificprocess.
EnzymeActivators
Enzymeactivityandinhibition
•
Coenzymescanbederivedfromsourcesotherthanvitamins:
• Chemicalsthathelptheenzymework.
• Activatorsincreasetheenzymereactionrate.
EnzymeInhibitors
• Chemicalsthatpreventtheenzymefromworking.
• Inhibitorsdecreasetheenzymereactionrate.
Active
Site
Activator
X
Binding
Site
Substrate
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EnzymeInhibitors
• Inhibitors(I) aremoleculesthatcausealossof
enzymeactivity
• Theypreventsubstratesfromfittingintothe
activesiteoftheenzyme:
E+SD ES® E+P
E+I D EI ® no Pformed
ReversibleInhibitors(Non-competitiveInhibition)
• Anon-competitiveinhibitor
hasastructurethatis
differentthanthatofthe
substrate
- itbindstoanallostericsite
ratherthantotheactivesite
- itdistortstheshapeofthe
enzyme,whichaltersthe
shapeoftheactivesiteand
preventsthebindingofthe
substrate
• Theeffectcannotbereversed
byaddingmoresubstrate,
buteventuallyreversedby
washingtheinhibitoraway.
SummaryofEnzymes-I
•
•
•
•
•
•
•
•
•
Enzymesaremostlyproteins
Theyarehighlyspecifictoareaction
Theycatalyzemanyreactionsincludingbreakingdownnutrients,storingand
releasingenergy,creatingnewmolecules,andcoordinatingbiological
reactions.
Enzymesuseanactivesite,butcanbeaffectedbybondingatotherareasof
theenzyme.
Someenzymesneedspecialmoleculescalledcofactorstocarryouttheir
function.
Cofactorsthatareorganicinnaturearecalledcoenzymes.
Coenzymesareusuallyderivedfromvitamins.
Coenzymestransferfunctionalgroupsfortheenzymetheyworkwith.
EnzymesareaffectedbychangesinpH,temperature,theamountof
substrate,cofactorsandinhibitors,aswellastheamountofallosteric
inhibitorsandactivatorsandconcentrationofproductsthatcontrolfeedback
inhibition.
Reversible Inhibitors (CompetitiveInhibition)
• Areversibleinhibitor goes
onandoff,allowingthe
enzymetoregainactivity
whentheinhibitorleaves
• Acompetitiveinhibitor is
reversibleandhasa
structurelikethesubstrate
- itcompeteswiththe
substratefortheactivesite
- itseffectisreversedby
increasingsubstrate
concentration
ExampleofaCompetitiveInhibitor
• Malonate isacompetitiveinhibitorofsuccinatedehydrogenase
- ithasastructurethatissimilartosuccinate
- inhibitioncanbereversedbyaddingsuccinate
IrreversibleInhibitors
• Anirreversibleinhibitor destroysenzymeactivity,usuallyby
bondingwithside-chaingroupsintheactivesite
http://upload.wikimedia.org/wikipedia/commons/thumb/6/6f/Major_digestive_enzymes.png/750px-Major_digestive_enzymes.png
Life Sciences-HHMI Outreach. Copyright 2009 President and Fellows of Harvard College
TheSixClasses
Biochemistry
3.2)Enzymes-II
Introductiontoenzymestructureand
function,andfactorsinvolvingtheir
actionsandpathways
Enzyme- Kinetics
•
•
•
•
•
•
EC1.Oxidoreductases
EC2.Transferases
EC3.Hydrolases
EC4.Lyases
EC5.Isomerases
EC6.Ligases
Additionalinformationonthesubclasses,thesub-subclassesandsub-subsubclasses(ie,fullenzymeclassificationandnames)canbefoundatthe
referencedweblink.
• FromtheWebversion,
http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html
Prof. Dr. Klaus Heese
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Some common types of enzymes
Hydrolases
Polymerases
Nucleases
Kinases
Proteases
Phosphatases
Synthases
ATP-ases
Isomerases
Oxidoreductases (dehydrogenases)
Enzyme Active Sites
Enzyme Active Sites
Active sites:
The region that binds substrate.
Only a small fraction of the enzyme.
Formed from AAs in different parts of the sequence.
Active sites:
Usually form a cleft or pocket.
Substrates are bound by
multiple weak interactions.
carboxypeptidase
pH,T
Lock-Key-model
Induced-Fit-Model
Properties of Enzymes
Reaction Thermodynamics
• Enzymes are highly efficient and specific
catalysts.
• Enzymes stabilize transition states.
How is Transition State Stabilization
Achieved?
• DGro is not changed
!
Enzymes stabilize the transition state, lowering the activation barrier.
How to Lower DG‡?
Enzymes organizes reactive groups into
proximity (---> induced-fit-model)
– acid-base catalysis: give and take protons
– covalent catalysis: change reaction paths
– metal ion catalysis: use redox cofactors, pKa shifters
• Uncatalyzed bimolecular reactions:
– electrostatic catalysis: preferential interactions with
• Uncatalyzed unimolecular reactions:
transition state
EA
• The enzyme lowers
the activation barrier
(EA or DG‡ )
compared to the
uncatalyzed
reaction.
• Enzymes alter rates, not equilibria.
• Reaction rates depend on concentrations of
enzymes, substrates, and on the efficiency
of the enzyme.
Rate
Acceleration
two free reactants ® single restricted transition state
conversion is entropically unfavorable
flexible reactant ® rigid transition state conversion is
entropically unfavorable for flexible reactants
• Catalyzed reactions:
Enzyme uses the binding energy of substrates to organize
the reactants to a fairly rigid ES complex
Entropy cost is paid during binding
Rigid reactant complex ® transition state conversion is entropically OK
EA
Enzymes stabilize the transition
state, lowering the activation barrier.
Enzyme Kinetics
Kinetics is the study of the rates of reactions
• Enzymes endow cells with the remarkable capacity to
exert kinetic control over thermodynamic potentiality
• Enzymes are the agents of metabolic function
• What we want to be able to determine:
– Maximum velocity
– Substrate affinity
– Inhibitor affinity
• What it can tell us:
– Flow through metabolic pathways
– Utilization of substrates
• What can we do with the information:
– Control and manipulate metabolic events
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Enzyme Kinetics
Keq =
Enzymatic Equations II
Enzymatic Equations
reminder: Consider:
[A] + [B]
Enzyme Kinetics
Enzyme Kinetics
Reaction Thermodynamics
[C] + [D]
E+S
ES
EP
E+S
E+S
E+P
There are at least three steps…….
Keq depends only on the nature of the products and the
reactants.
E+P
E+P
Km = (k-1+ kcat )/k1
usually kcat <<< k-1 , so Km = k-1 /k1 = Kd
The Michaelis-Menten Equation
Kinetics: Vmax and Km
Important Conclusions of Michaels Menten Kinetics
kcat
Vmax = the maximum reaction rate.
Km = substrate concentration where V = Vmax /2
Measures affinity of enzyme for substrate.
• when [S] >> KM, the equation reduces to
• when [S] << KM, the equation reduces to
The Michaelis-Menten equation
• Ideal Rate:
Enzyme Kinetics
Reaction rate (V) varies with substrate
concentration.
Effect of Substrate Concentration
• when [S]= KM, the equation reduces to
Enzyme Kinetics
Enzyme Kinetics
ES
kcat
=[E][S]=(k-1+kcat )/k1 [ES]--->with[E]total=[E]t and[S]0 =[S]attimepoint0:
[E]=[E]t –[ES]--->([E]t –[ES])[S]=Km [ES]--->[ES]=[S][E]/Km +[S]--->
istheproductformationcontrollingthevelocityofthereaction:--->
V0 =kcat [ES]=kcat {[S][E]/Km +[S]};if[S]highsothatenzymefullysatisfied:Vmax =kcat [E]t
--->V0 =Vmax [S]/Km +[S]
Reaction will proceed spontaneously only when the change
in free energy (DG) is negative.
V = Vmax
k-1
EP
atsteadystate:forward=reversereaction:d[ES]/dt =-d[ES]/dt <--->k1 [E][S]=k-1+kcat [ES]
[C][D]
[A][B]
[S]
Km + [S]
ES
k1
v=
Vmax [ S ]
Km + S
• Deviations due to:
– Limitation of
measurements
– Substrate inhibition
– Substrate prep
contains inhibitors
– Enzyme prep
contains inhibitors
Effect of Substrate Concentration
Vmax and Km
Different substrates, Vmax and Km
Enzyme Kinetics
Vmax [S]
V= K--------------M +[S]
Enzyme Kinetics
Enzyme Kinetics
Vmax
Vmax dependsontheamountofenzyme.
Km is a property of both enzyme and substrate.
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• A plot of 1/V versus 1/[S] will give a straight line
with slope of KM/Vmax and y intercept of 1/Vmax
• Such a plot is known as a Lineweaver-Burk double
reciprocal plot
v0
1
v0
1
v0
=
=
([S]+Km )
Vmax [S]
=
Enzyme inhibitors are important for a variety of reasons
3) they can be used to gain information about the regulation
or control of a metabolic pathway.
4) they can be very important in drug design.
The“normal” wayanenzymefunctionsiswhenthespecific
substratebindstotheactivesiteandcreatestheproducts.
•
Asimilarsubstratecanalsobondtotheactivesitecovalently
andirreversibly.Thispreventstheenzymefromfunctioning.
Irreversibleinhibition.
•
Asimilarsubstratecanbindtotheactivesite,notpermanently,
andpreventsthedesiredsubstratefromenteringtheactivesite.
Thischangestheproductsandfunctioningoftheenzyme.Thisis
calledcompetitiveinhibition.
•
Amoleculecanbondtoanotherpartoftheenzymeandcausea
changeinconformation.Thischangecausestheactivesiteto
changeshapeaswell.Thischangeinshapepreventsthedesired
substratefromenteringtheactivesite.Thisiscallednoncompetitiveinhibition.
=
[S]
Vmax [S]
Km 1
Vmax [S]
+
+
Km
Vmax [S]
1
Vmax
V and [S] can be
determined
experimentally
Enzyme inhibition
Tool to study enzymatic reactions.
Important in host/pathogen interactions.
Important in drug design.
Irreversible (suicide) inhibition (eg - nerve gas).
Reversible inhibition:
competitive (eg. - transition state analogues).
non-competitive.
uncompetitive.
Type of inhibition can be determined experimentally.
EnzymeActivators
Enzymeactivityandinhibition
•
Vmax [S]
([S]+Km )
Linearized double-reciprocal
plot is good for analysis of
two-substrate data or
inhibition.
Lineweaver – Burk Double
Reciprocal Plots
Enzyme Inhibition
1) they can be used to gain information about the shape on
the enzyme active site and the amino acid residues in the
active site.
2) they can be used to gain information about the chemical
mechanism.
Significance of Km
Determination of Kinetic Parameters
• Chemicalsthathelptheenzymework.
• Activatorsincreasetheenzymereactionrate.
Enzyme Kinetics
Enzyme Kinetics
Lineweaver – Burk Double Reciprocal Plots
• It is difficult to determine Vmax experimentally
• The equation for a hyperbola can be transformed into
the equation for a straight line by taking the
reciprocal of each side
• The formula for a straight line is y = mx + b
• Km is a constant
• Small Km means tight binding; high Km means weak
binding
• Useful to compare Km for different substrates for one
enzyme
Hexokinase : D-fructose – 1.5 mM
D-glucose – 0.15 mM
• Useful to compare Km for a common substrate used
by several enzymes
Hexokinase: D-glucose – 0.15 mM
Glucokinase: D-glucose – 20 mM
Inhibition Patterns
Inhibitors act in a variety of mechanisms
• An inhibitor may bind at the same site as one of the
substrates
– these inhibitors structurally resemble the substrate
• An inhibitor may bind at an alternate site affecting
catalytic activity without affecting substrate binding
• Many inhibitors do both
• Most common types
– Competitive
– Mixed or Non-competitive
– Uncompetitive
EnzymeInhibitors
• Chemicalsthatpreventtheenzymefromworking.
• Inhibitorsdecreasetheenzymereactionrate.
Active
Site
Activator
X
Binding
Site
Substrate
10
9/16/16
EnzymeInhibitors
• Inhibitors(I) aremoleculesthatcausealossof
enzymeactivity
• Theypreventsubstratesfromfittingintothe
activesiteoftheenzyme:
E+SD ES® E+P
E+I D EI ® no Pformed
Competitive Inhibition
ReversibleInhibitors(CompetitiveInhibition)
• Areversibleinhibitor goes
onandoff,allowingthe
enzymetoregainactivity
whentheinhibitorleaves
• Acompetitiveinhibitor is
reversibleandhasa
structurelikethesubstrate
- itcompeteswiththe
substratefortheactivesite
- itseffectisreversedby
increasingsubstrate
concentration
Competitive Inhibition
ExampleofaCompetitiveInhibitor
• Malonate isacompetitiveinhibitorofsuccinatedehydrogenase
- ithasastructurethatissimilartosuccinate
- inhibitioncanbereversedbyaddingsuccinate
Competitive Inhibition
• Unimolecular
Reaction
• A competitive inhibitor reduces the amount of
free enzyme available for substrate binding
thus increasing the Km for the substrate
• Bimolecular
• The effect of a competitive inhibitor can be
overcome with high concentrations of the
substrate
ReversibleInhibitors(Non-competitiveInhibition)
• Anon-competitiveinhibitor
hasastructurethatisdifferent
thanthatofthesubstrate
- itbindstoanallostericsite
ratherthantotheactivesite
- itdistortstheshapeofthe
enzyme,whichaltersthe
shapeoftheactivesiteand
preventsthebindingofthe
substrate
• Theeffectcannotbereversed
byaddingmoresubstrate,but
eventuallyreversedbywashing
theinhibitoraway.
Reaction
Change of Lineweaver – Burk graph
by enzyme inhibition
Mixed or Non-Competitive Inhibition
Non-Competitive
• The inhibitor can bind to both free enzyme and the ES
complex
• The affinity of the inhibitor to the two complexes might be
different
– If binding of inhibitor changes the affinity for the substrate, Km will be
changed and called mixed inhibition
– If only Vmax affected called Non-competitive inhibitor
Change of Lineweaver – Burk graph
by enzyme inhibition
11
9/16/16
Mixed Inhibition
Mixed Inhibition
•
•
The result will be decrease in
Vmax and either an increase or
decrease in Km
The effect of an non-competitive
inhibitor can only be partially
overcome by high concentrations
of the substrate
Uncompetitive Inhibition
Uncompetitive
Reactionoftheirreversibleinhibitor
diisopropylfluorophosphate (DFP)witha
serineprotease;
DFPisairreversiblecholinesteraseinhibitor:
Diisopropyl fluorophosphate isaverypotent
neurotoxin.ItsLD50inratsis1.3mg/kg.It
combineswiththeaminoacidserineatthe
activesiteoftheenzyme
acetylcholinesterase, anenzymethat
deactivatestheneurotransmitter
acetylcholine.Neurotransmittersareneeded
tocontinuethepassageofnerveimpulses
fromoneneurontoanother(ortothe
(striated)muscle)acrossthesynapse.Once
theimpulsehasbeentransmitted,
acetylcholinesterase functionstodeactivate
theacetylcholinealmostimmediatelyby
breakingitdown.Iftheenzymeisinhibited,
acetylcholineaccumulatesandnerve
impulsescannotbestopped,causing
prolongedmusclecontraction.Paralysis
occursanddeathmayresultsincethe
respiratorymusclesareaffected.
Uncompetitive Inhibition
• An uncompetitive
inhibitor binds to the
enzyme substrate
complex but not to
free enzyme
• The result is a
decrease in Vmax
and Km
• The effect of an
uncompetitive
inhibitor can not be
overcome by high
concentrations of the
substrate
IrreversibleInhibitors
• Anirreversibleinhibitor destroysenzymeactivity,usuallyby
bondingwithside-chaingroupsintheactivesite
SummaryofEnzymes-II
•
•
•
•
•
•
•
•
•
Enzymesaremostlyproteins
Theyarehighlyspecifictoareaction
Theycatalyzemanyreactionsincludingbreakingdownnutrients,storingand
releasingenergy,creatingnewmolecules,andcoordinatingbiological
reactions.
Enzymesuseanactivesite,butcanbeaffectedbybondingatotherareasof
theenzyme.
Someenzymesneedspecialmoleculescalledcofactorstocarryouttheir
function.
Cofactorsthatareorganicinnaturearecalledcoenzymes.
Coenzymesareusuallyderivedfromvitamins.
Coenzymestransferfunctionalgroupsfortheenzymetheyworkwith.
EnzymesareaffectedbychangesinpH,temperature,theamountof
substrate,cofactorsandvarioustypesofinhibitors.
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