enzymes: properties and activitiesenzymes • enzymes are biological catalysts. • the majority of...

Enzymes:PropertiesandActivities

Dr. Sameh Sarray Hlaoui

Lecture9-10

Enzymes• Enzymesarebiologicalcatalysts.

• Themajorityofenzymesareproteins,butsomeconsistofRNAoracomplexofproteinandRNA(ribozymes)

• Theyincreaseratesofreactionsunderphysiologicalconditions(temperatureandpH)withoutthemselvesbeingusedupinthereaction.

• Nearlyall(thousands)reactionsinourbodiesarecatalyzedbyenzymesconvertingasubstrate toaproduct

Enzymes• Namingenzymes:

– Startingwiththenameofthesubstrate,then– thetypeofreactionandendingwith– Enditwiththesuffix–ase

• Thetertiarystructureoftheenzymeformsaspecialpocketknownastheactivesite

• Substrate:moleculeuponwhichtheenzymeacts).Thisbindstothissite,formtheenzyme-substratecomplex

E+S ⇄ ES⇄ E+P

ClassificationofEnzymes

• Hundredsofreactionsinourcellsarecatalyzed byenzymes

• AreclassifiedintoSIX classesaccordingtothetypeofreactiontheycatalyze:• Oxidoreductases• Transferases• Hydrolyses• Lyases• Isomerases• ligases

Enzymes• Enzymesarespecific,actononeorfewtypesofmolecules(enzymesubstrates)togivesometypeofmolecularproduct.

• Thisbindingofthesubstrateforcestheenzymetochangeconformation (oxygenbindingtoHb)

• Thisdecreasestheenergyofactivation neededtochangethesubstratetoatransitionstate (unstable)andthatallowsthereactiontoproceedtoformaproductandregeneratetheenzyme

Enzymes• Transitionstate:intermediateformedbetweentheinitialchemicalreactantandtheproduct

• Enzymesreducethefreeenergyofactivationofthereactionstheycatalyze.

• Forsubstrate(reactant)tobecomeproduct(catalyzed ornot),thesubstratemustpassthroughthetransitionstate.

Chemicalreactions

§ Enzymesmake reactions gofaster:§ Byincreasingthetemperature(itmakesmoleculesmovefaster),however,biologicalsystemsareverysensitivetotemperaturechanges.

§ Enzymescanincreasetherateofreactionswithoutincreasingthetemperature:Theydothisbyloweringtheactivationenergy

§ Theycreateanewreactionpathway “ashortcut”

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.

Cofactors• Anadditionalnon-proteinmoleculethatisneededbysomeenzymestohelpthereaction

• Tightly(covalently)boundcofactorsarecalledprostheticgroups.Thesearenotreleasedaftercompletionofthereaction

• Cofactorsthatareboundandreleasedeasily(reversiblebinding)arecalledcoenzymes.

Nitrogenase enzyme with Fe, Mo and ADP cofactors

• Bindingofapoenzyme tocofactor producesaholoenzyme

Coenzymes• Apoenzyme:Anenzymethatrequiresacofactor (anon-proteinmolecule)toparticipateinthereaction

• Bindingofapoenzyme tocofactorproducesaholoenzyme

• Somecofactorsare:– Metalions: thesehelpstabilizetheES,– Organicmolecules:alsocalledcoenzymes andarederivedfromvitamins.

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

Coenzymes• TrueCoenzymes:

– Bindreversibly totheenzyme.– Releasedafterthecatalyticreactioniscomplete.

• Prostheticgroups– Coenzymesthatarecovalentlyboundtotheenzyme.

– Thesearenotreleasedaftercompletionofthereaction.

Factors affecting Enzymes

• Substrate concentration[S]• pH• Temperature• Inhibitors

FactorsAffectingEnzymeReactionVelocity

• Substrateconcentration [S]– Increasing[S]increasestherateofthereaction(firstorder)hyperbolicallyinproportionwiththeincreasein[S]untilallactivesitesaresaturated

• Thereactionreachesitsmaximalvelocity(Vmax)andfurtheradditionofsubstratehasnoeffectonthereactionrate,i.e.thereactionisnolongerdependenton[S](zeroorder)– Itisunderzeroorderconditionsthatenzymeactivityisnormallymeasured

Orderofreaction

§ When[S]isgreaterthanKm,thevelocityisconstantandequaltoVmax.Therateofreactionthendependsonthesubstrateconcentration(theenzymeissaturatedwithsubstrate),andissaidtobezeroorder.

§ When[S]islessthanKm,thevelocityofreactionisproportionaltothesubstrateconcentration.Therateofreactionissaidtobefirstorder.

Effectof[S]The[S]atwhichthereactionrateishalfVmaxisaconstantfortheparticularenzymeandsubstrate,andisknownastheKm (Michaelis-Menten constant)

Itisameasureoftheaffinity oftheenzymefortheparticularsubstrate.

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)

Effectof[S]• WhenitisdifficulttodetermineVmax andhencetheKmofareactionfromtheaboveplot,youshouldplotthereciprocal of[S]againstthereciprocalofthevelocity(1/v against1/[S])

• Thischangesthehyperboliccurveintoastraightline,whichinterceptsthex-axisat-1/Km andthey-axisat1/Vmax.

• ThisplotisknownasLineweaver-Burkeplot

EffectofpH• AchangeinpH

– Alterstheionizationstateofionisable residues

– Thisoccursonbothenzymesurfaceandintheactivesite

• ThereactionratechangestoreachmaximumatenzymeoptimalpH.

• FurtherchangesinpHmaydenature theenzyme,leadingtodecreasedrate

EffectofTemperature• Increasingtemperatureincreasestherateofthereactiontoamaximumknownastheoptimumtemperature

• Abovethistemperaturetheenzymeproteinisdenaturedandthereactionratedecreases

• Ifapersondevelopsfever,allenzymeswillhavehigherreactionvelocities

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 :

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 .

EnzymeInhibitionAcompetitiveinhibitoris:• Structurallysimilartothesubstrate.

• Competeswiththesubstrateforbindingtheenzymeactivesite.

• Athighsubstrateconcentration[S]orlowinhibitorconcentration[I],theinhibitorwillcauselittledisruptiontonormalenzymefunction

CompetitiveEnzymeInhibition

• EffectonVmax:theeffectoftheinhibitorisreversedbyincreasing[S].Athighconcentrationof[S],thereactionvelocityreachtheVmax observedintheabsenceofinhibitor

• EffectonKm:theinhibitorincreasestheKm.Thismeansthatinthepresenceofcompetitiveinhibitor,moresubstrateisneededtoachieve1/2Vmax

• InLinweaver–Burk plot:TheKm increaseswhiletheVmax remainsunchanged.

Noncompetitive EnzymeInhibition

• EffectonVmax:noncompetitiveinhibitioncannotbeovercomebyincreasingthe[S],thereforeitdecreasestheVmax ofthereaction

• Effect onKm:noncompetitivedonotinterferewiththebindingofsubstratetoenzyme.ThereforetheenzymeshowsthesameKminthepresenceorintheabsenceofcompetitiveinhibitor

• InLinweaver–Burkplot:TheVmaxdecreasesinthepresenceofinhibitorandKmisunchanged

RegulationofEnzymeActivity

Essentialprocesstocoordinatenumerousmetabolicprocess:

Ø Regulationofallosteric enzymes

Ø Regulationofenzymesbycovalentmodifications

RegulationofEnzymeActivityAllostericsites

• Someenzymeshavebindingsitesotherthantheactivesite

• Whenaneffector bindstothatallostericsite,theresultingchangeinconformationoftheenzymemayincrease(+)ordecrease(-)thereactionvelocityorthesubstrateconcentrationthatgiveshalfVmax

EnzymeActivity• Theeffectormoleculemaybetheendproductofametabolicpathway

• Thismaycausenegativefeedback(feedbackinhibition)tostoptheproductionofmoreofthisendproduct

Regulationofenzymesbycovalentmodifications

• Manyenzymesareregulatedbycovalentmodifications.

• Mostoftenbytheadditionorremovalofphosphategroupsfromspecificaminoacids(serine,threonine,tyrosine),whichcanbe:• Active(stimulationofenzyme)• Inactive(inhibitionofenzyme

activity).• Bindingamoleculecovalentlytoan

enzymewillalteritsconformation,andthereforeitsactivity.

• Theresponseisusuallyveryfast(acute)

Induction&RepressionofEnzymeSynthesis

• Thepreviousmechanismsofregulatingenzymeactivitydonotinvolveanychangeintheamountofenzymeprotein.

• Cellsmayregulatetheamountofenzyme(andthereforethetotalactivity,Vmax)bychangingtherateofsynthesisoftheenzymeprotein.

• Thisoccursatthelevelofthegenebyinduction(stimulation)orrepression (inhibition)

• Itisaslowprocess(hours– days)