1

LactateDehydrogenaseCharacterizationAngelaKC

Introduction: AtneutralpH,theenzymelactatedehydrogenase(LDH)catalysestheoxidationofNADHcoupled,highlyexergonicandstereospecificreductionofpyruvatetoL‐lactateasshowninthereactionbelow(NelsonandCox.2008)

LDHisabisubstrateenzymeconsistingofNAD+asthehydrogencarryingcoenzymeandeitherlactateorpyruvateasthesecondsubstrate.LDHisatetramerofMr140,000thatconsistsofcombinationsoftwosimilarpolypeptides,namedMandH,formuscleandheartrespectively.ThesetwotypesofpolypeptidesmakeupdifferentcombinationstomaketheLDHtetramer,M4,M3H,M2H2,MH3andH4.ThesevariouscombinationsarecalledtheisozymesofLDH(Zubay.1988).ThevariousisozymesofLDHhavedifferentfunctionsdependingonthenatureofthetissuetheyarelocatedin.TheM4isozymeinthemuscleshowsalesserdegreeofLDHinhibitioninthisanaerobictissuebytheincreasedconcentrationsofpyruvate.Whereas,theH4isozymeintheaerobichearttissuesshowagreaterdegreeofLDHinhibitionthroughincreasesinpyruvateconcentrations.Thus,theenzymeactivityisdependentontheisozymetype(Zubay.1988). Tocharacterizetheseisozymes,theirkineticswerestudiedexperimentallytodeterminedifferencesintheirMichaelisconsonant(Km)andmaximumvelocity(Vmax)valuesandcomparethemtostandardliteraturevalues.EnzymeassaysofvaryingsubstrateconcentrationswerepreparedtoevaluatethereconstitutedrabbitM4isozyme.Threekineticsplots,Michaelis‐Menten,Lineweaver‐BurkeandEadie‐HofsteeoftheresultingabsorbancesweremadeandcomparedtothatfromtheplotsmadefromthegivenH4absorbancedata. FurtherexplorationofLDHkineticswascarriedoutbystudyingthechangeinenzymeassayabsorbancesataconstantpyruvateconcentrationupontheadditionofvariousconcentrationsofthepesticideKepone,whichisaknownLDHinhibitor.ItcompetitivelyinhibitsLDHactionatconcentrationaslowas0.01mM(HendricksonandBowden.1975). SDS‐PAGEwasusedtofindouttheMsubunitmolecularweightoftheM4isozymethroughthecomparisonoftheelectrophoreticmobilityviaRfvaluesofthesubunittothatofthestandardproteinmasses.SizeexclusionchromatographybyHPLCwasthenusedtodeterminethemolecularweightoftheisozymethroughtheuseofastandardcurve.Thecomparisonofthevaluesof

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theweightsforthesubunitandthetotalproteinmasswasexpectedtoreiteratethefactthattheLDHisindeedatetramer. MolecularmodellingwasthenusedtodeterminethedistancesbetweenthevariousresiduesandsubstratesintheactivesitetoverifytheproposedLDHcatalysismechanism.ThestructureusedforthisanalysiswastheMsubunitoftheM4isozymeobtainedfromProteinDataBankoftheBrookhavenNationalLab. Withtheseanalyticaltools,itwasassumedthatLDHM4isozymethathadbeenisolatedpreviouslyandpurifiedcouldbecharacterizedtoinordertounderstandtheenzymeactionmore.ExperimentalProcedures:A.Reconstitutionoflyophilizedsample TheisolatedLDHsamples4+5,diluted10XwiththeΔA340/minof0.1689waslyophilizedtoconserveenzymeactivityduringstorage.Thevolumeofthesample4+5was1.41mL.Giventhedilutionfactorsandtheabsorbances,theenzymeactivitywascalculatedtobe65.25unitsofenzyme/mL(KC.2011). Afterstorage,theenzymewasrestoredbyaddinghalftheoriginalvolumestothesample,705μLofdeionizedwaterforsample4+5aweekpriortoactualusetoallowallofthesolidifiedmassestogointosolution.Vortexingorshakingwasavoidedtoconserveenzymestructuresandthusenzymefunction. Duringtheexperiment,halfofthevolumeofthereconstitutedsamplewastaken,i.e.,353.5μLof4+5andanequalvolumeofthedeionizedwaterwasaddedintothissampletogettotheoriginalconcentration.Thesamplewasthendiluted1:10anditsenzymeassaywasconductedtorecorditsΔA340/mintoensurethattheactivityhadnotchanged.TheΔA340/minofthereconstituted4+5samplewas0.1326,whichgivestheenzymeactivityof63.96units/mL(KC.2011)

Thus,sample4+5wasusedtoconductthekineticsexperiments.

B.Kinetics ThereconstitutedLDHM4sample4+5,withΔA340/minof0.1326wasusedtoconductanenzymeassaywithvaryingconcentrationsofpyruvatepresentinthem.TheabsorbancesmeasuredweretobeusedtoplottheMichealis‐Menten,Lineweaver‐BurkeandEadie‐HofsteeplotsandthuscalculatetheVmaxandKmvaluesfromeachoftheseplots.TheMichaelis‐Mentenplotisalsousedtodescribewhetherthereissubstrate(pyruvate)inhibitionpresentinthisisozymeornot.Table1liststhevolumesofsubstrate,enzymeandLDHusedtoperformtheassay.

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Table1:Thevolumesofreactantsintheenzymeassay(3mL)

[Pyruvate] in assay (mM)

Vol of 22.7mM Pyruvate (µL)

Vol of KPhos buffer, pH 7 (µL)

Vol of NADH (5mg/mL) (µL)

Vol of LDH (µL)

0.025 3.30 2936.7 50 10 0.050 6.60 2933.4 50 10 0.10 13.2 2926.8 50 10 0.25 33.0 2907.0 50 10 0.50 66.1 2873.9 50 10 1.0 132 2807.8 50 10 2.5 330 2609.6 50 10 5.0 661 2279.2 50 10 TheVmaxandKmvaluesforthemuscleLDHisozymewerethen

comparedwiththatofthepre‐obtaineddatafortheheartLDHisozymeusingthethreeabove‐mentionedplotstoaccountfordifferences. Theexplorationkineticsstudywasextendedbyobservingtheeffectsofthepesticidekepone(chlordecone)onLDHactivity.Aconstantpyruvateconcentrationof0.10mMwasusedtoconductanenzymeassayagainstincreasingkeponeconcentrations.Theabsorbancedataobtainedfromtheassayswereplottedtogiveavelocityversuskeponeconcentrationgraphtodeterminethetypeofinhibitionpresentedbykepone.

Fig1:ThestructureofKeponeTable2:ThevolumesofreactantsintheenzymeassayforKepone(3mL)

Tube

Kphos buffer, pH 7, (mL)

95% ethanol

(µL) Pyruvate

(mM)

22.7mM pyruvate

(µL) [Kepone]

Vol of 0.01M

Kepone (µL)

Vol NADH (5mg/mL)

(µL)

1 2.937 0 0.1 13.2 0 0 50 2 2.637 300 0.1 13.2 0 0 50 3 2.637 298.5 0.1 13.2 0.005 1.5 50 4 2.637 297 0.1 13.2 0.01 3 50 5 2.637 292.5 0.1 13.2 0.025 7.5 50 6 2.637 285 0.1 13.2 0.05 15 50 7 2.637 270 0.1 13.2 0.1 30 50 8 2.637 225 0.1 13.2 0.25 75 50 9 2.637 150 0.1 13.2 0.5 150 50 10 2.637 0 0.1 13.2 1 300 50

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C.Molecularweightdetermination1.SDSGelElectrophoresis SDS‐PAGEwasperformedonthepuresample4+5tocalculatethevalueofthesubunitweightofMbycomparingtheelectrophoreticmobilityofthepuresamplebandtothatofthestandardproteinsamples.SDS‐PAGE(sodiumdodecylsulfatepolyacrylamidegelelectrophoresis)separatesproteinsubunitsbasedontheirsize,whichcanthenprovideuswithinformationabouttheirmolecularweight.SDSisananionicdetergentthatgivestheproteinauniformnegativechargebycoveringit,sotheysubunitsmovetowardstheanodethroughthegel,therebyseparatingthroughtheirmass.SDSisabletodosobydissociatingthenativeproteinsintotheirlinearformsofsubunitswiththehelpofreducingagentslikedithiothreotol(DTT)thatdisruptsthedisulfidebridges.Theacrylamidegelusedactsasaporoussievethatletsthesmallermoleculestravelfasterthanthelargerones,thusthebandsthattravelthefarthestinthegelarethelighterproteinmasses. Thecrude,dialyzedandpuresampleswereruninthisSDS‐PAGEalongwithsigmaLDH,NEBiolabproteinstandardsandbovineserumalbumin(BSA)at~140V.A3XreducingsampleSDSsamplebuffer(RSB)containing10μLof30Xreducingagent,DTTto100μLof3XSDSsamplebufferthatcontainsbromphenolblue,trackingdye,SDSandglyceroltomakethesampleheavier.Thegelwasloadedinthesequenceshownintable2.Table3:Thevolumesofsamples,waterandRSBaddedineachlaneinthegel.

Lane

Sample volume (µL) RSB (µL)

Deionized water (µL)

1 Blank - 5 -

2 Crude (~1mg/mL) 1 10 19

3 Sigma LDH 6 10 14

4 NE Standard I 20 - -

5 Pure (4+5) I 20 - 10

6 BSA (1mg/mL) 4 10 16

7 Pure (4+5)II 20 - -

8 NE Standard II 25 10 -

9 ASPD (~1m/mL) 1 10 19

10 Blank - 5 - About23μLofeachsamplewasloadedintotheirrespectivelanesandthegelwasrununtilthedyewasabout0.5cmfromthebottomofthegel.ThegelwasthenstainedwithCoomassiebluestainforabouthalfanhour,rinsedwithwaterandthendestainedtwicefor15minutesforeachdestainingprocess.Thegelwasthenphotographedandthebandsineachlanemeasuredforthedistancesthattheyhadtraveledfromthewells.

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TheNEstandardshadtheirBSAandtriosephosphateisomerase(TPI)presentindoubletheconcentrationthanotherstandardproteins,becauseitisexpectedthatthemolecularweightofthesubunitwillbewithinthisrangeanditiseasiertodeterminetheidentityofeachbandthroughlane6BSA.Throughthemeasurementofthedistancesthattheproteinunitsseenasbandshavetraveledandthetotaldistancetraveledbythedyeitself,anRfcalculationwasconductedusingtheformula,Rf=(distanceofproteinmigration)/(distanceoftrackingdyemigration).AstandardcurvewasthengeneratedusingthetwoNEstandardRfdatatoextrapolatetheweightofLDHsubunitusingtheequationgeneratedfromthelineofbestfitofthelogofstandardmolecularweightversusRfplot.ThiswasexpectedtogivethesubunitmolecularmassforM.2.SizeexclusionchromatographybyHPLC DataforsizeexclusionchromatographywasobtainedinordertodeterminethetotalmolecularweightoftheLDHtetramer,andtoconfirmthefactthattheenzymeisindeedatetramer.Sizeexclusionchromatographyorgelfiltrationchromatographyiscanseparatemoleculesofdifferentsizesthroughtheirdifferentialdiffusionintothegelpores.Thelargermoleculesdonotentertheporesofthepackinggelunlikethesmallerones,andareelutedfasterthroughthefluidvolumeasaresult. Thus,thesmallermoleculesspendmoretimepassingthroughthestationaryphasepackingmaterialofsphericalgelbeadsofhydrophilicbondedphasesilicainthecolumn;whilethelargemoleculestraveleasilythroughthemobilephase.HPLCwasusedtospeedupthiselutionprocesssothatmicroliterquantitiesofproteininjectedintothecolumnwouldelutethroughthecolumnfasterandbedetectedbytheUVdetectorat280nm. 20μLofstandardLDHsamplewasinjectedintothecolumnandtheabsorbancewasrecordedfor15minutes.Thesamewasdonewith20μLofLDHsample.ThestandardplotoflogofmolecularweightsversusretentiontimeofthegivenstandardproteinmasseswereusedtocalculatethemolecularweightofLDHthroughthegivendataforLDHretentiontime. ThemolecularmassforLDHtetramerobtainedfromthischromatographycanbecomparedwiththatoftheMsubunitobtainedfromSDS‐PAGEtoconfirmthatLDHisinfactatetramer.Table3.Showsthedataobtainedforthestandardproteinsize‐exclusionchromatography.Table4:HPLCproteinstandarddata

Protein Rt/min Molecular

weight

Log of molecular

weight

Thyroglobulin 7.28 670,000 5.82607 Bovine gammaglobulin 8.018 158,000 5.19866 Rabbit ovalbumin 8.938 44,000 4.64345 Bovine myoglobulin 9.56 17,000 4.23045 Cyanocobalamine 11.738 1,350 3.13033

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D.Molecularmodeling TheMpolypeptidechainofLDHM4isozymefromBrookhavenNationalLabobtainedviaProteinDataBankwasusedtostudythepossiblemechanismofactionofLDHusingmolecularmodeling.ThestructureofLDHwasstudiedandthedistancebetweenaminoacidresiduesandsubstratesweremeasuredtounderstandthemodeofLDHaction. Theflexibleloopthatenfoldsthesubstrateandcoenzymeintheactivesite,madeofresidues#98‐120,wasidentifiedandeachoftheaminoacidwaslisteddown.DistancesbetweenothervariousactivesiteresiduesandfunctionalgroupspresentinthecoenzymeNADHandthesubstrateanalogoxamatewerealsomeasuredtostudytheinteractionswithintheactivesite.Thesubstrateanalogoxamatewasusedinsteadofpyruvatetoarresttheenzymetopreventfurtherconformationalchangesthatleadtowardsproductrelease.

Fig2:PyruvateandoxamatestructuresResultsandDiscussion:A.Reconstitutionoflyophilizedsample Priortolyophilisation,theΔA340/minforsample4+5was0.1689fora1:10dilutedsample.Thus,givingtheenzymeactivityof81.45unitsofenzyme/mL.Whenthislyophilizedsamplewasreconstitutedbyadditionofdeionizedwater,theΔA340/minobtainedforthesamedilutionwas0.1326,whichgavetheenzymeactivityof63.96units/mL.Thepercentageofactivityrecoveredafterreconstitutionwas78.53%. Itshowsthatalthoughlyophilisationisausefultechniquetostoreandrecoverenzymeactivityitisnotacompleteactivityconservationtechnique.Aspreservingtheproteinsinastableformisnecessaryfortheirefficacy,variousoptimizationmethodsinadditiontolyophilisationshouldbecarriedout.Anexperimentshowedthatpolyethyleneglycol(PEG)andlactosehadastabilizingeffectontheLDHduringthescreening.Theexperimentalsoshowedthatthetemperatureoffreezingandratesofsublimationalsoaffectedtherelativestabilityoftheproteinstructure(Grantet.al.2009).AnotherexperimentshowedthatadditionofPEG8000successfullyrecovered90%ofenzymeactivityafterLDHreconstitution,unlikethe60‐80%standardactivityrecoveryofLDHafterlyophilization(MiandWood.2004).Thus,asthepercentagerecoveryfallsundertherange,thesimplelyophilizationwasconsideredtobesuccessfulenoughinthiscase.

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B.Kinetics FromtheanalysisofkineticsofLDHM4isozymeofrabbitmuscle,thefollowinggraphsforMichaelis‐Meten,Lineweaver‐BurkeandEadie‐Hofsteewereprepared.SimilargraphsweremadeforthepreobtaineddatafortheH4rabbitisozymeofLDH.TheKmandVmaxwerecalculatedfromeachofthesegraphsforthetwoisozymesandlistedundertable5.

0.00000

0.00500

0.01000

0.01500

0.02000

0.02500

0 1 2 3 4 5

VasD[NADH]/min(mM/m

in)

[pyruvate]mM

Graph1:MichaelisMentenformuscleLDH(M4)Km

Vmax

8

y=4.9884x+29.417R²=0.99868

0.00

50.00

100.00

150.00

200.00

250.00

0 5 10 15 20 25 30 35 40 45

1/V

1/[S]mM­1

Graph2:Lineweaver­BurkeLDHmuscle(M4)

y=‐0.1417x+0.03R²=0.98538

0.00000

0.00500

0.01000

0.01500

0.02000

0.02500

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2

mMNADH/m

in

V/[S]

Graph3:Eadie­HofsteeMuscleLDH(M4)

9

0.00000

0.00500

0.01000

0.01500

0.02000

0.02500

0.03000

0 1 2 3 4 5 6 7 8 9 10 11

VasD[NADH]/min

[pyruvate]mM

Graph4:MichaelisMentenforLDHheart(H4)

Vmax

Km

10

y=0.3774x+35.148R²=0.99707

0

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30

1/VmM­1min

1/[pyruvate]mM­1

Graph5:LineweaverLDHheart(H4)

y=‐0.0107x+0.0284R²=0.99577

0

0.005

0.01

0.015

0.02

0.025

0.03

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

mMNADH/m

in

v/[S]min­1

Graph6:Eadie­HofsteeLDHHeart(H4)

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Table5:AcomparativekineticsdatafordifferentplotsforthetwoisozymesofLDH

Michaelis-Menten

Lineweaver-Burke Eadie-Hofstee

Muscle LDH V max (mM/min) 0.023 0.034 0.030

Km (mM) 0.09864 0.1696 0.1417

Heart LDH V max (mM/min) 0.028 0.028 0.028

Km (mM) 0.0114 0.0107 0.0107 EarlierstudiesshowthattheKmvaluesfortheM4andH4LDHisozymeswereobservedtobe0.35mMand0.10mMrespectively(StambaughandBuckley.1967).Theexperimentalvalueshoweverweremuchlowerthantheexpectedliteraturevaluessuggestingthattheenzymewasmoreactivethanexpected.TheaverageKmforM4isozymeis0.1038mMcomparedtothe0.35mMandthatforH4is0.0107mMcomparedto0.10mM.Thismighthavebeenthecasebecausetheenzymeactivesitemighthavebeenmoreaccessibleduringthestructuraldestabilizationthatoccurredduringandafterlyophilization. LiketheLDHheartdata,LDHmusclefollowedMichaelis‐Mentenkineticsfromthepyruvateconcentrationsof0.025mMto0.50mMafterwhichitshowedtheeffectsofnegativefeedbackgeneratedduetothelargerproductionoftheproductlactate.Thisshowstheregulatorymechanismsthatarepresentinthemetaboliccyclesinlivingbeingsbywhichpyruvateinhibitsthedehydrogenase(Zubay.1988). Similarly,heartLDHdataalsofollowedMichaelis‐Mentenkineticsuntil0.513mM.Afterthatconcentrationofpyruvate,thecurveslopesdownwardinsteadofreachingthesaturatedplateaulevelduetofeedbackmechanismsinducedbylactateformation. Theseisozymeshavedifferentkinetics,astheyarepresentindifferenttissuetypesthathavedifferentenergyrequirementsandlevelofpyruvateproductionanduse.Themuscleisactiveandhenceanaerobic,soproducesalotofpyruvatethatisconvertedintolactate.Thus,M4showslesssubstrateinhibitionthanH4isozyme.TheheartLDHispresentintheaerobichearttissues,andmostofthepyruvategoesintoKreb’scycletoproducemoreenergy,andhenceLDHmorereadilyinhibitedbythepyruvate(Zubay.1988). TheanalysisofLDHM4kineticsinpresenceofthepesticidekeponeisgivenbelow.Table6:DataforthechangeinvelocityofthereactionwithincreasingKeponeconcentrations

[Kepone] mM ΔA/min

V in mM/min

0 0.0919 0.01477 0.0050 0.0682 0.01096 0.010 0.0659 0.01059 0.025 0.0436 0.00701 0.050 0.0223 0.00359 0.100 0.0177 0.00285

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Fromgraph7itisseenthatKeponecompetitivelyinhibitstheenzymeLDHatconcentrationsevenaslowas0.005mM.AlthoughstructurallyKeponeisdistinctlydifferentfrompyruvate,itsuccessfullypreventstheoxidationofNADHbyLDHmuscleisozyme.IncreasingKeponeconcentrationsisalsoseentodrasticallyreducetheenzymeactivityduetothecompetitivenatureoftheinhibitioninduced.C.Molecularweightdetermination1.SDSGelElectrophoresis

TheresultsfromSDS‐PAGEandsizeexclusionchromatographybyHPLCwereusedtodeterminethetotalmolecularweightoftheLDHenzymeaswellastheeachofthesubunitweightsfortheenzyme.SDS‐PAGEwasconductedandastandardcurvewasgeneratedtocalculatethemassofeachoftheLDHsubunits.SizeexclusionchromatographybyHPLCwasconductedtocalculatethetotalmolecularweightoftheLDHenzymeandindoingsoitwasusedtodeterminethattheenzymeconsistsoffoursubunitsasanticipated. ThesubunitweightLDHM4isozymewasexpectedtobe35,000DaasdescribedbyearlierSDS‐PAGEanalysis(Javed,et.al.1995).Anotherstudysuggeststhatthesubunitmolecularweightisconservedamongdifferentspecies(FosmireandTimasheff.1972). DuringtheanalysisoftheSDS‐PAGEdata,theBSAsamplehadRfof0.2222(1.0cm)and0.4667(2.10cm),sothe0.2222RfwasomittedinboththestandardandtheBSAdata.Thus,thestandardcurveofgraph8andtheequationofthelinegeneratedwereusedtocalculatethesubunitweightofMfortheLDHmuscleM4isozymeas34,906Da.

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0 0.02 0.04 0.06 0.08 0.1

VinmM/m

in

[Kepone]mM

Graph7:Effectofincreasing[Kepone]onLDHactivity

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Table7:Thedistancetravelledbyeachbandinthelanes3­8andthecalculatedRfDye migration = 4.5cm

Sigma LDH/cm

Standard 1/cm

Pure LDH/cm BSA/ cm

Standard 2/cm

Pure LDH/cm

3.3 0.8 3.3 1 0.8 3.3 3.5 1 3.5 2.1 1 3.5 1.2 1.2 1.4 1.45 1.6 1.6 2.1 2.1 2.45 2.5 2.9 2.9 3.5 3.5 4.15 4.15

Dye migration = 4.5cm, Rf = band migration/dye migration

Sigma LDH Rf

Standard 1 Rf

Pure LDH Rf BSA Rf

Standard Rf Pure Rf

0.7333 0.1778 0.7333 0.2222 0.1778 0.7333 0.7778 0.2222 0.7778 0.4667 0.2222 0.7778

0.2667 0.2667 0.3111 0.3222 0.3556 0.3556 0.4667 0.4667 0.5444 0.5556 0.6444 0.6444 0.7778 0.7778 0.9222 0.9222

Figure3:TheSDS­PAGEgelforLDHmigrationwithstandards

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2.SizeexclusionchromatographybyHPLC

y=‐0.8579x+5.2102R²=0.98908

0.0000

1.0000

2.0000

3.0000

4.0000

5.0000

6.0000

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

LogofMW

Rf

Graph8:ThestandardSDS­PAGEcurveshowingthelineofbestfitfromBSAtoTPI

y=‐0.5936x+10.012R²=0.98942

0.00000

1.00000

2.00000

3.00000

4.00000

5.00000

6.00000

7.00000

7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12

Logofmolecularweight

Retentiontime(Rt)inmin

Graph9:StandardcurveforsizeexclusionchromatographybyHPLC

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Figure4:ThepeaksforsizeexclusionchromatographybyHPLC

AsthesizeexclusionchromatographybyHPLCcalculationsgivesthetotal

molecularweightofLDH(4subunit)tobe140,605Da,theRfvalueof0.7778withthesubunitmolecularweightof34,906Daandtheonewith0.7333,giving38,114.5mustbetheaccuratemigrationbandsoftheLDHsubunits.AsthebandwiththeRf0.7778wasmuchdarkerthantheoneat0.7333,itcanbeassumedthattheheaviervariantofthesubunitispresentatalowerconcentrationthanthelighterone.Thus,theLDHsubunitofmolecularmass34,906Dacanbe

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regardedasthevalueofeachoftheLDHsubunitmass,giving4X34,905=139620DaastheanticipatedtotalLDHmasswhichiscomparableto140,605Dafromsizeexclusiondata.PreviousstudiesgivethetotalmolecularweightofLDHas142,000Da(FosmireandTinasheff.1972).

Hence,itcanbeassumedthatbothoftheexperimentalprocedureswereusedefficientlytocalculatethetotalmolecularweightandsubunitmolecularweightforLDH.D.Molecularmodeling TheresiduesoftheactivesitewerelabeledanddeterminedviaProteinDataBankviewerfortheM4LDHisozymeaslistedintable12.TheM4structureofinterestwastheMpolypeptidechainwithNADHandthepyruvateanalog,oxamate.ThestructureandthemodeofactionoftheactivesiteoneachofthesubunitsofLDHareknowntobeconservedwhiletheaminoacidresidesareknowntobedifferentamongdifferentLDHsamples.IthasaboundNADHinitsactivesiteasthecoenzymethatisoxidizedtoNAD+duringtheconversionofpyruvatetolactateinareactionthatisstronglyfavouredintheforwarddirection.Inthisreactionthecentralcarbonofpyruvate(‐C=O)isknowntoundergochangefromsp2conformationtoansp3conformationoflactate.

Fig5:NAD+ TheLDHenzymeactivesiteisknowntobindtheNADHbeforethebindingoftheactualsubstratepyruvate.TheC‐4atomofthenicotinamideringisaccessibletoassociationswiththereactivecarbonofpyruvate.AstheenzymebindstheNADH,itundergoesconformationchangevisiblethroughtheassociationoftheadeninepartoftheringandAsp53.Thedistancewasfoundtobe3.526ÅforAsp52andtheribosepartoftheadenineringshowingthatthebindingofNADHtotheenzymebringstheconformationalchange,pullingtheaminoacidresiduesclosertotheNADH.Similarly,Arg101formsanionpairwiththepyrophosphategroupofNADH.Thedistancebetween–NH2ofArg99andthenearestoxygensofthephosphateinNADHwasfoundtobe2.978Åand

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4.706Å.Thisionpairformationisregardedasthemainassociationinthemobileloopthatbringsaboutfurtherconformationalchangesintheactivesitethatpromotestheenzymeactivityonpyruvate.Themobileloopmovementfunctionsalidwithtwohingesthatclosesdowntheactivesiteandbeginstheenzymaticreactions.Themovementofthemobileloopsbringsaboutaconformationalchangeofaround11Å.Also,Asp30isknowntoholdtheNADHinplace. Intheactivesite,Tyr85isknowntoactwiththeadenineoftheNADH.Thedistancemeasuredherewas4.006ÅforTyr83andofthe–OHgroupinTyrandtheN,nextto–NH2intheadeninering.ShowingthatthereisindeedacloseinteractionbetweenthetwothatisnotasstrongastheothermobileloopinteractionswithNADH. Arg171isknowntoholdthepyruvateinplacethroughassociationwiththecarboxylicacidorpyruvate.Here,apyruvateanalog,oxamateisusedtoarresttheenzymeinapositionwithitsmobileloopclosedbutwithouttheoccurrenceofanyforwardreactionthatleadstoproductformation.Arg169–NH2wasfoundtobe3.141Åand2.797Åfromtheoxygen’softheCOO‐partofoxamate. AssociationofArg109–NH2withthecentralcarbonyloxygenofpyruvatewasalsoanindicatorofthesubstratebeinginassociationwiththeactivesite.ThedistancebetweenArg106andthecentraloxygenwasfoundtobe2.866Å. His195Nintheringandthecentralcarbonyloxygenofpyruvatewerealsoknowntointeractduringtheenzymeaction.Thedistancewasmeasuredtobe2.702ÅfromHis193. TherewasalsoassociationseenbetweentheNADHandthepyruvateanalogasshownbythedistancebetweentheC4ofthenicotinamideringofNADHandthecentralcarbonofoxamate.Thedistancewasfoundtobe3.201Å. Asthesubstrate‐bindingpocketliesdeepintheenzymeactivesite,makingthesiteaccessibletopyruvateisthusdonethroughtheassociationofNADHwiththemobileloopresiduesthatallowthepyruvatetofirstenterandthehingetheactivesitecloseoncethepyruvateisheldbytheactivesiteresidues.IntheactivesitethepyruvateisinassociationwiththecatalyticallyimportantHis195andArg109thatstabilizethepolartransitionstates.Arg171inthesitesolvatesthenegativelychargedcarboxylgroupofpyruvate. AssociationofNADHwiththeenzymeactivatesthemobileloop(residues98‐110)causestheclosingoftheloopovertheactivesiteandArg109isbroughtincloseassociationwiththesubstrate,whilewaterisexpelledandpyruvateandNADHarebroughtclosetogethertoformanassociationthatleadstocatalysisandlactateformation(McClendonet.al.2005). Themeasureddistancesbetweenactivesiteandmobileloopresidueswiththecoenzymeandthesubstrateareallwithin2‐4ÅindicatingthatthereareindeedassociationsamongtheresiduesandtheNADHandpyruvatealongwiththeassociationofNADHandpyruvatethemselves.

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Fig6:TheactivesiteoftheenzymeLDHwithboundoxamateConclusion: Theexperimentalprocedurefollowedasequentialcharacterizationoftherabbitmuscle(M4)isozyme.Althoughnotoptimized,thereconstitutionofthelyophilizedsamplegavea78.53%recoveredsamplethatwasconsideredasthepuresampleofLDHwithanactivityof63.96unitsofenzyme/mL. Thedataforkineticstudiesofthetwoisozymes,M4andH4wereconductedusingenzymeassaysandthreekineticcurves,Michealis‐Menten,Eadie‐HofsteeandLineweaverBurke.TheaverageKmforM4wasfoundtobe0.1038mMandforH4was0.0107mM.ThevaluesforKmweremuchlowerthananticipated,whichmightbethepossibleresultofdestabilityduringnotoptimizedlyophilizationproceduresthatmaketheactivesitemoreaccessibletothesubstrateorduethefluctuationsinpHandtemperature(PlaceandPowers.1984). Keponeontheotherhand,asanticipatedshowedkineticsofcompetitiveinhibition,startingatconcentrationsaslowas0.005mM. SDS‐PAGEandsizeexclusionchromatographygavethesubunitmolecularweightandtotaltetramerweightofofLDHM4tobe34,906Daand140,605Darespectively.Theseexperimentaldatawereconsistentwithpriorobtaineddata. Throughmolecularmodelingtheenzymewasseentoefficientlytransferahydrideionfromthepro‐RfaceofthereducednicotinamidegroupofNADHtothecentralcarbonylcarbonofpyruvatetoturnittolactate.Initialassociationoftheenzymeactivesitewasseentobepresentbeforetheassociationofthe

19

substratewiththeactivesiteresidues.Thedistancesmeasuredwerewithin4ÅandshowedthatTyr85,Asp53andArg101holdtheNADHinplace,while,Arg169,Arg109andHis195heldthepyruvateanalog.TherewasalsoanassociationobservedbetweentheNADHandthepyruvate.Thus,themobileloopwasseentoundergoconformationalchangesthatenclosedtheenzymeactivesiteandbroughtthecoenzymeandthesubstrateclosetogethertoundergocatalysis.References:Fosmire,G.andTimasheff,S.N.1972.Molecularweightofbeefheartlactate

dehydrogenase.Biochemistry.11(13):2455‐2460Grant,Y.,MatejtschuckP.andDalby,P.A.2009.Rapidoptimizationofprotein

freeze‐dryingformulationsusingultrascale‐downandfactorialdesignofexperimentinmicroplates.BiotechnologyandEngineering.105(5):957‐964

Hendrickson,C.M.andBowden,J.A.1975.Theinvitroinhibitionofrabbitmuscle lactatedehydrogenasebyMirexandKepone.J.Agric.FoodChem.23(3):

407‐409Javed,M.,Yousuf,F.A.,Hussain,A.N.,Ishaq,M.andWaqar,M.A.1995.

PurificationandpropertiesoflactatedehydrogenasefromliverofUromastixhardwickii.Comp.Biochem.Physiol.111B(1):27‐34.

KC,Angela.2011.Isolationandpurificationoftheenzymelactatedehydrogenase.3‐9.

McClendon,S.,Zhadin,N.andCallender,R.2005.TheapproachtotheMichaeliscomplexinlactatedehydrogenase:Thesubstratebindingpathway.BiophysJ.89(3):2024‐2032

Mi,Y.andWood,G.2004.Theapplicationandmechanismsofpolyethyleneglycol8000onstabilizinglactatedehydrogenaseduringlyophilization.PDAJ.Pharm.Sci.Technol.58(4):192‐202

Place,A.R.andPowers,D.A.1984.Purificationandcharacterizationofthelactatedehydrogenase(LDH‐B4)allozymesofFundulusheteroclitus.J.Biol.Chem.259(2):1299‐1308

Place,A.R.andPowers,D.A.1984.Kineticcharacterizationofthelactatedehydrogenase(LDH‐B4)allozymesofFundulusheteroclitus.J.Biol.Chem.259(2):1309‐1318

Sevnic,A.,Sari,R.andFadillioglu,E.2005.Theutilityoflactatedehydrogenaseisoenzymepatterninthediagnosticevaluationofmalignantandnonmalignantascites.JNatlMedAssoc.97(1):79‐84.

Stambaugh,R.andBuckley,J.1967.TheenzymicandmolecularnatureoflacticdehydrogenasesubbandsandX4isozyme.J.ofBio.Chem.242(18):‐4059

Zubay,G.1988.Biochemistry.2Supportinginformation:A.Calculationsforenzymeactivityforsample4+5Pre‐lyophilization:ΔA340/min=0.1689

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Dilutionfactor=10XCorrectedΔA340/min=1.689ΔA/Δt=εb(Δc/Δt)1.689=6.22X10^3XΔcΔc=2.715X10^‐4Mmin‐1LDHinassay=2.715X10^‐4X3.00X10^‐3=8.415μmolmin‐1LDHenzymeactivity=81.45units/mLPost‐reconstitution:ΔA340/min=0.1326Dilutionfactor=10XCorrectedΔA340/min=1.326ΔA/Δt=εb(Δc/Δt)1.326=6.22X10^3XΔcΔc=2.132X10^‐4Mmin‐1LDHinassay=2.132X10^‐4X3.00X10^‐3=6.396μmolmin‐1LDHenzymeactivity=63.96units/mLPercentageofLDHactivityrecovered:(63.96/81.45)X100%=78.53%B.DataandcalculationsforkineticsTable8:ThedataforLDHmuscleisozyme

[S] mM 1/[S]

(mM-1) ΔA/min V as

ΔNADH/min 1/V V/[S] 0.025 40 0.027 0.00434 230.37 0.1736 0.050 20 0.049 0.00788 126.94 0.1576 0.10 10 0.081 0.01302 76.790 0.1302 0.25 4 0.118 0.01897 52.712 0.0759 0.50 2 0.144 0.02315 43.194 0.0463 1.0 1 0.141 0.02267 44.113 0.0227 2.5 0.4 0.124 0.01994 50.161 0.0080 5.0 0.2 0.106 0.01704 58.679 0.0034

ForMichaelis­MentenofmuscleLDH,V=Vmax[S]/[S]+KmFromthegraph,Vmax=0.02315mM/min~0.023mM/minForKm,V=0.01302mM/minand[S]=0.10mMV=Vmax[S]/[S]+Km0.01302=0.02315X0.10/(0.10+Km)0.01302Km+0.01302X0.10=0.02315X0.100.01302Km=0.001302Therefore,Km=0.09864mMForLineweaver­BurkedataofmuscleLDHTheequationofthelineofbestfitisy=4.9884x+29.417correspondingtotheequationofLineweaver‐Burkeplot,1/V=(1/Vmax)+(Km/VmaxX1/[S])

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Therefore,y‐intercept=1/VmaxSlope=Km/Vmax=4.9884X‐intercept=‐1/KmLet,y=0=4.9884x+29.417‐29.417=4.9884xOr,x‐intercept=‐1/Km=‐5.897mM‐1Or,Km=0.1696mMSlope=4.9884=0.1696/VmaxOr,Vmax=0.033999mM/min~0.034mM/minForEadie­HofsteeMuscleLDHTheequationofthelineofbestfitis,y=­0.1417x+0.03,where,V=(‐KmXV)/[S]+VmaxSlope=‐Km=‐0.1417Or,Km=0.1417mMYintercept=Vmax=0.03mM/minTable9:ThedataforLDHheartisozyme

[S] mM 1/[S]

(mM-1) ΔA/min V as

ΔNADH/min 1/V V/[S] 0.036 27.778 0.136 0.02186 45.735 0.60736 0.055 18.182 0.148 0.02379 42.027 0.43262 0.092 10.870 0.160 0.02572 38.875 0.27960 0.256 3.9063 0.169 0.02717 36.805 0.10613 0.513 1.9493 0.173 0.02781 35.954 0.05422 1.03 0.9709 0.140 0.02251 44.429 0.02185 2.49 0.4016 0.113 0.01817 55.044 0.00730 4.97 0.2012 0.093 0.01495 66.882 0.00301 9.98 0.1002 0.084 0.01350 74.048 0.00135

ForMichaelis­MentenofheartLDH,V=Vmax[S]/[S]+KmFromthegraph,Vmax=0.02781mM/min~0.028mM/minForKm,V=0.02379mM/minand[S]=0.055mMV=Vmax[S]/[S]+Km0.02379=0.02781X0.055/(0.055+Km)0.02379Km+0.02379X0.55=0.02871X0.0550.02379Km=0.000271Therefore,Km=0.011375mM~0.0114mMForLineweaver­BurkedataofheartLDHTheequationofthelineofbestfitisy=0.3774x+35.148correspondingtotheequationofLineweaver‐Burkeplot,1/V=(1/Vmax)+(Km/VmaxX1/[S])Therefore,y‐intercept=1/VmaxSlope=Km/Vmax=0.3774X‐intercept=‐1/KmLet,y=0=0.3774x+35.148‐35.148=0.3774x

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Or,x‐intercept=‐1/Km=‐93.132mM‐1Or,Km=0.0107mMSlope=0.3774=0.0107/VmaxOr,Vmax=0.02835mM/min~0.028mM/minForEadie­HofsteeheartLDHTheequationofthelineofbestfitisy=­0.0107x+0.0284,where,V=(‐KmXV)/[S]+VmaxSlope=‐Km=‐0.0107Or,Km=0.0107mMYintercept=Vmax=0.0284mM/minB.DataandcalculationsformolecularweightdeterminationTable10:ThecalculatedlogofmolecularweightandRfforstandardproteinsamples

Standard protein

Molecular weight (Da) Log of MW Rf

Myosin 212,000 5.3263 0.1778

MBP-b-galactosidase 158,194 5.1992 0.2667

B-galactosidase 116,351 5.0658 0.3167 Phosphorylase B 97,184 4.9876 0.3556 BSA 66,409 4.8222 0.4667

Glutamic dehydrogenase 55,561 4.7448 0.5500 Maltose binding 42,710 4.6305 0.6444 Thioredoxin reductase 34,622 4.5394 0.7778 Triosephos Isomerase 26,972 4.4309 0.9222 Fromthegraph8,theequationofthelineofbestfitis,y=‐0.8579x+5.2102,wherey=logofMWandx=RfGiventhatRfforLDHare0.7333and0.7778,itispossibletocalculatethelogoftheLDHsubunitMWandhencethemolecularweight.YorlogsubunitMW(3.30cm)=‐0.8579X0.7333+5.2102=4.5811LDHsubunitMW=38,115.4DaYorlogsubunitMW(3.50cm)=‐0.8579X0.7778+5.2102=4.5429LDHsubunitMW=34,906Da

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Table11:ThelogoftheMWandRfforsizeexclusionstandards

Protein standards Rt/min

Molecular weight

Log of molecular

weight

Thyroglobulin 7.28 670,000 5.82607

Bovine gammaglobulin1 8.018 158,000 5.19866 Rabbit ovalbumin 8.938 44,000 4.64345 Bovine myoglobulin 9.56 17,000 4.23045 Cyanocobalamine 11.738 1,350 3.13033

FromthesizeexclusionchromatographybyHPLC,theretentiontime(Rt)fortheLDHsamplewasfoundtobe8.194min.

Fromthestandardcurve,theequationofthelinewasobtained,y=‐0.5936x+10.012,wherey=logofmolecularweightandx=retention

time.Hence,y=logofLDHmolecularweight=‐0.5936X8.194+10.012=

5.148Or,molecularweightofLDH=10^5.148=140,605Da.

Table12:TheaminoacidresiduesoftheLDHactivesiteResidue #

Amino acid

Residue #

Amino acid

98 Ala 109 Leu 99 Arg 110 Val 100 Gln 112 Gln 101 Gln 113 Arg 102 Glu 114 Val 103 Gly 115 Asn 104 Glu 116 Ile 105 Ser 117 Phe 106 Arg 118 Lys 107 Leu 119 Phe 108 Asn 120 Ile