Enzymes: Properties and ActivitiesEnzymes • Enzymes are biological catalysts. • The majority of...
Transcript of Enzymes: Properties and ActivitiesEnzymes • Enzymes are biological catalysts. • The majority of...
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Enzymes:PropertiesandActivities
Dr. Sameh Sarray Hlaoui
Lecture9-10
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Enzymes• Enzymesarebiologicalcatalysts.
• Themajorityofenzymesareproteins,butsomeconsistofRNAoracomplexofproteinandRNA(ribozymes)
• Theyincreaseratesofreactionsunderphysiologicalconditions(temperatureandpH)withoutthemselvesbeingusedupinthereaction.
• Nearlyall(thousands)reactionsinourbodiesarecatalyzedbyenzymesconvertingasubstrate toaproduct
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Enzymes• Namingenzymes:
– Startingwiththenameofthesubstrate,then– thetypeofreactionandendingwith– Enditwiththesuffix–ase
• Thetertiarystructureoftheenzymeformsaspecialpocketknownastheactivesite
• Substrate:moleculeuponwhichtheenzymeacts).Thisbindstothissite,formtheenzyme-substratecomplex
E+S ⇄ ES⇄ E+P
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ClassificationofEnzymes
• Hundredsofreactionsinourcellsarecatalyzed byenzymes
• AreclassifiedintoSIX classesaccordingtothetypeofreactiontheycatalyze:• Oxidoreductases• Transferases• Hydrolyses• Lyases• Isomerases• ligases
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Enzymes• Enzymesarespecific,actononeorfewtypesofmolecules(enzymesubstrates)togivesometypeofmolecularproduct.
• Thisbindingofthesubstrateforcestheenzymetochangeconformation (oxygenbindingtoHb)
• Thisdecreasestheenergyofactivation neededtochangethesubstratetoatransitionstate (unstable)andthatallowsthereactiontoproceedtoformaproductandregeneratetheenzyme
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Enzymes• Transitionstate:intermediateformedbetweentheinitialchemicalreactantandtheproduct
• Enzymesreducethefreeenergyofactivationofthereactionstheycatalyze.
• Forsubstrate(reactant)tobecomeproduct(catalyzed ornot),thesubstratemustpassthroughthetransitionstate.
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Chemicalreactions
§ Enzymesmake reactions gofaster:§ Byincreasingthetemperature(itmakesmoleculesmovefaster),however,biologicalsystemsareverysensitivetotemperaturechanges.
§ Enzymescanincreasetherateofreactionswithoutincreasingthetemperature:Theydothisbyloweringtheactivationenergy
§ Theycreateanewreactionpathway “ashortcut”
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Enzymes• Enzymesmayhave:
– Absolutespecificity:actonaspecificsubstrate– Broadspecificity:actongroupofrelatedsubstrates
• Isoenzymes: enzymesthat catalyzethesamereactionbuthavedifferentphysicalproperties,(e.g.aminoacidorsubunitcomposition).
• Example: Creatine Kinase(CK)consist of2subunitswhichcanbeeitherB(braintype)orM(muscletype):therefore,thereare3isoenzymes:
– CK-BB isoenzyme:expressedinbrain– CK-MM inskeletalmuscle– CK-MB specificallyinheartmuscle.
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Cofactors• Anadditionalnon-proteinmoleculethatisneededbysomeenzymestohelpthereaction
• Tightly(covalently)boundcofactorsarecalledprostheticgroups.Thesearenotreleasedaftercompletionofthereaction
• Cofactorsthatareboundandreleasedeasily(reversiblebinding)arecalledcoenzymes.
Nitrogenase enzyme with Fe, Mo and ADP cofactors
• Bindingofapoenzyme tocofactor producesaholoenzyme
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Coenzymes• Apoenzyme:Anenzymethatrequiresacofactor (anon-proteinmolecule)toparticipateinthereaction
• Bindingofapoenzyme tocofactorproducesaholoenzyme
• Somecofactorsare:– Metalions: thesehelpstabilizetheES,– Organicmolecules:alsocalledcoenzymes andarederivedfromvitamins.
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CoenzymesCoenzymeorProstheticGroup Vitamin Reactions
Lipoicacid Lipoicacid Hydrogenandacylgroupacceptor
Tetrahydrofolic acid(THF) Folicacid Onecarbontransfer
Biotin Biotin Carboxylation
ThiaminPyrophosphate(TPP) Thiamine B1 Oxidativedecarboxylation
FlavinMononucleotide(FMN) Riboflavin B2 Oxidation/Reduction
FlavinAdenineDinucleotide(FAD)
Riboflavin B2 Oxidation/Reduction
NicotinamideAdenine Dinucleotide(NAD+)
Niacin B3 Oxidation/Reduction
NicotinamideAdenineDinucleotidePhosphate(NADP+)
Niacin B3 Oxidation/Reduction
CoenzymeA(CoA) Pantothenicacid
B5 Acetyloracylgrouptransfer
Pyridoxalphosphate(PALP) Pyridoxine B6 Aminotransferases,aminoaciddecarboxylases,racemases
Cobamide Cobalamin B12 Onecarbontransfer
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Coenzymes• TrueCoenzymes:
– Bindreversibly totheenzyme.– Releasedafterthecatalyticreactioniscomplete.
• Prostheticgroups– Coenzymesthatarecovalentlyboundtotheenzyme.
– Thesearenotreleasedaftercompletionofthereaction.
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Factors affecting Enzymes
• Substrate concentration[S]• pH• Temperature• Inhibitors
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FactorsAffectingEnzymeReactionVelocity
• Substrateconcentration [S]– Increasing[S]increasestherateofthereaction(firstorder)hyperbolicallyinproportionwiththeincreasein[S]untilallactivesitesaresaturated
• Thereactionreachesitsmaximalvelocity(Vmax)andfurtheradditionofsubstratehasnoeffectonthereactionrate,i.e.thereactionisnolongerdependenton[S](zeroorder)– Itisunderzeroorderconditionsthatenzymeactivityisnormallymeasured
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Orderofreaction
§ When[S]isgreaterthanKm,thevelocityisconstantandequaltoVmax.Therateofreactionthendependsonthesubstrateconcentration(theenzymeissaturatedwithsubstrate),andissaidtobezeroorder.
§ When[S]islessthanKm,thevelocityofreactionisproportionaltothesubstrateconcentration.Therateofreactionissaidtobefirstorder.
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Effectof[S]The[S]atwhichthereactionrateishalfVmaxisaconstantfortheparticularenzymeandsubstrate,andisknownastheKm (Michaelis-Menten constant)
Itisameasureoftheaffinity oftheenzymefortheparticularsubstrate.
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Effectof[S]• The[S]atwhichthereactionrateishalf Vmax (1/2Vmax)isaconstant knownastheKm (Michaelis-Menten constant)
•Michaelis-Menten equation:describeshowreactionvelocityvarieswith[S]
V0=initialreactionvelocityVmax=maximalvelocityKm=Michaelis constant[S]=substrateconcentration
Km:reflects theaffinity oftheenzymeforthatsubstrate:
- LowKmreflectsahighaffinityoftheenzymeforthesubstrateand
- ConverselyforlargeKm(lowaffinitybecauseahighconcentrationofsubstrateisneededtohalfsaturateheenzyme)
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Effectof[S]• WhenitisdifficulttodetermineVmax andhencetheKmofareactionfromtheaboveplot,youshouldplotthereciprocal of[S]againstthereciprocalofthevelocity(1/v against1/[S])
• Thischangesthehyperboliccurveintoastraightline,whichinterceptsthex-axisat-1/Km andthey-axisat1/Vmax.
• ThisplotisknownasLineweaver-Burkeplot
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EffectofpH• AchangeinpH
– Alterstheionizationstateofionisable residues
– Thisoccursonbothenzymesurfaceandintheactivesite
• ThereactionratechangestoreachmaximumatenzymeoptimalpH.
• FurtherchangesinpHmaydenature theenzyme,leadingtodecreasedrate
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EffectofTemperature• Increasingtemperatureincreasestherateofthereactiontoamaximumknownastheoptimumtemperature
• Abovethistemperaturetheenzymeproteinisdenaturedandthereactionratedecreases
• Ifapersondevelopsfever,allenzymeswillhavehigherreactionvelocities
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Effectof[S](cont)• WhenitisdifficulttodetermineVmax andhence
theKm ofareactionfromtheplot,youshouldplotthereciprocal of[S]againstthereciprocalofthevelocity(1/v against1/[S])
• Thischangesthehyperboliccurveintoastraightline, whichinterceptsthex-axisat-1/Km andthey-axisat1/Vmax.
• ThisplotisknownasLineweaver-Burkeplot(doublereciprocalplot).ItcanbeusedtodetermineKm andVmax aswellastodeterminethemechanismofactionofinhibitors
• TheequationofLineweaver-Burke :
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EnzymeInhibition• Enzymeinhibitors:molecules&factorsthat
interactwiththeenzyme,andpreventitfromworking.Classifiedinto:
• NonspecificInhibitors:Theseeffectallenzymesinthesameway.
• SpecificInhibitors:Theseexerttheireffectsonasingleenzyme.Theyareclassifiedinto:– Reversible:theinhibitorcandissociatefrom
theenzyme,andcatalyticactivityisregained:• Competitive:withrespectofthesubstrate
• Non-competitive:donotresembletheenzyme'ssubstrate,thusdonotbindenzymeattheactivesitebutatothersiteandinducingenzymetoundergoconformationalchanges.
– Irreversible: whentheenzymeispermanentlyinactivated .
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EnzymeInhibitionAcompetitiveinhibitoris:• Structurallysimilartothesubstrate.
• Competeswiththesubstrateforbindingtheenzymeactivesite.
• Athighsubstrateconcentration[S]orlowinhibitorconcentration[I],theinhibitorwillcauselittledisruptiontonormalenzymefunction
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CompetitiveEnzymeInhibition
• EffectonVmax:theeffectoftheinhibitorisreversedbyincreasing[S].Athighconcentrationof[S],thereactionvelocityreachtheVmax observedintheabsenceofinhibitor
• EffectonKm:theinhibitorincreasestheKm.Thismeansthatinthepresenceofcompetitiveinhibitor,moresubstrateisneededtoachieve1/2Vmax
• InLinweaver–Burk plot:TheKm increaseswhiletheVmax remainsunchanged.
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Noncompetitive EnzymeInhibition
• EffectonVmax:noncompetitiveinhibitioncannotbeovercomebyincreasingthe[S],thereforeitdecreasestheVmax ofthereaction
• Effect onKm:noncompetitivedonotinterferewiththebindingofsubstratetoenzyme.ThereforetheenzymeshowsthesameKminthepresenceorintheabsenceofcompetitiveinhibitor
• InLinweaver–Burkplot:TheVmaxdecreasesinthepresenceofinhibitorandKmisunchanged
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RegulationofEnzymeActivity
Essentialprocesstocoordinatenumerousmetabolicprocess:
Ø Regulationofallosteric enzymes
Ø Regulationofenzymesbycovalentmodifications
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RegulationofEnzymeActivityAllostericsites
• Someenzymeshavebindingsitesotherthantheactivesite
• Whenaneffector bindstothatallostericsite,theresultingchangeinconformationoftheenzymemayincrease(+)ordecrease(-)thereactionvelocityorthesubstrateconcentrationthatgiveshalfVmax
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EnzymeActivity• Theeffectormoleculemaybetheendproductofametabolicpathway
• Thismaycausenegativefeedback(feedbackinhibition)tostoptheproductionofmoreofthisendproduct
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Regulationofenzymesbycovalentmodifications
• Manyenzymesareregulatedbycovalentmodifications.
• Mostoftenbytheadditionorremovalofphosphategroupsfromspecificaminoacids(serine,threonine,tyrosine),whichcanbe:• Active(stimulationofenzyme)• Inactive(inhibitionofenzyme
activity).• Bindingamoleculecovalentlytoan
enzymewillalteritsconformation,andthereforeitsactivity.
• Theresponseisusuallyveryfast(acute)
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Induction&RepressionofEnzymeSynthesis
• Thepreviousmechanismsofregulatingenzymeactivitydonotinvolveanychangeintheamountofenzymeprotein.
• Cellsmayregulatetheamountofenzyme(andthereforethetotalactivity,Vmax)bychangingtherateofsynthesisoftheenzymeprotein.
• Thisoccursatthelevelofthegenebyinduction(stimulation)orrepression (inhibition)
• Itisaslowprocess(hours– days)