Download - Phosphofructokinase (PFK) Exercise - STARstar.mit.edu/.../biochem/exercises/pfk_exercise_ver5.pdf · 2012-08-29 · Ver. 5 ‐ D. Sinha and L. Alemán 1 Phosphofructokinase (PFK)

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Ver.5‐D.SinhaandL.Alemán

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Phosphofructokinase(PFK)ExerciseLearningObjectivesInthisexercise,youwilluseStarBiochem,aprotein3Dviewer,toexplorethestructureandfunctionoftheenzymephosphofructokinase(PFK).BackgroundLivingorganisms,bothunicellularandmulticellular,requireenergytoliveandfunction.Energyisstoredandprovidedintheformofadenosinetriphosphate(ATP).ATPisproducedbymultiplecellularprocessesincludingglycolysis,aswellasaerobicandanaerobicrespiration.PFKisthemostimportantregulatoryenzymeofglycolysis.Itirreversiblycatalyzestherate‐limitingstepofglycolysis:theconversionoffructose‐6‐phosphateandATPtofructose‐1,6‐diphosphateandADP.

Fructose6‐phosphate fructose1,6‐biphosphate

PFKexistsasahomotetramerinbacteriaandmammals.EachofthefourmonomersofPFKiscomposedoftwosimilardomains.OnedomainhasanATPbindingpocket.Theotherdomaincontainsthesubstrate‐bindingsiteandtheallostericsite.Anallostericsiteisaregulatorybindingsitethataffectsenzymeactivityandisdistinctfromthesubstrate‐bindingsiteofanenzyme.VariousactivatorsandinhibitorsregulatePFK.ExamplesofPFKactivatorsareADP,AMPandfructose2,6diphosphate.ExamplesofPFKinhibitorsareATP,phosphoglycerate(PGA)andcitrate.

ADP fructose2,6diphosphate PGA ATPGettingstartedwithStarBiochem• TobeingusingStarBiochem,pleasenavigateto:http://web.mit.edu/star/biochem.• ClickontheStartbuttontolaunchtheapplication.• ClickTrustwhenapromptappearsaskingifyoutrustthecertificate.• UnderFile,clickonOpen/Importandselect“2PFK”andclickOpen.

YouarenowviewingthestructureofPFK(2PFK)witheachbondintheproteindrawnasaline(“bondsonly”view).YouwillseetwoindependentPFKstructureswithinthe2PFKfile.Thisisanartifactofhowthestructurewasdetermined.Pleasefocusononeofthetwostructureswhileansweringthequestionsinthisexercise.

ATPH++ADP

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Practicechangingtheviewpointofthisproteinintheviewwindow:

Mac PCTOROTATE

clickanddragthemouse left‐clickanddragthemouse

TOMOVEUP/DOWNRIGHT/LEFT

apple‐clickanddragthemouse right‐clickanddragthemouse

TOZOOM

option‐clickanddragthemouse Alt‐left‐clickanddragthemouse

TakeamomenttolookatthestructureofPFK(2PFK)fromvariousanglesinthis"bondsonly"view.Beforeproceedingtoanswerthequestions,youcanreviewthebasicstructuresandtermsonthenextpagewhichyoucanrefertoduringthisexercise.

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PROTEINSTRUCTUREBASICSEachproteinhasthefollowingthreelevelsofproteinstructure:PrimarystructureListstheaminoacidsthatmakeupaprotein’ssequence,butdoesnotdescribeitsshape.SecondarystructureDescribesregionsoflocalfoldingthatformaspecificshape,likeahelix,asheet,oracoil.TertiarystructureDescribestheentirefoldedshapeofawholeproteinchain.Inaddition,someproteinsinteractwiththemselvesorwithotherproteinstoformlargerproteinstructures.HowtheseproteinsinteractandfoldtoformalargerproteincomplexistermedQuaternarystructure.

CHEMICALSTRUCTURESOFTHEAMINOACIDSThe20aminoacidsshareacommonbackboneandaredistinguishedbydifferent‘R’groups,highlightedinvariouscolorsbelow.

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ProteinStructureQuestions1HowmanyaminoacidscomprisePFK(2PFK)?• ClickonStructure.• ClickonPrimarywhichshowstheaminoacidsthataresequentiallyjoinedthroughpeptidebondstomaketheamino/polypeptidechain.Theaminoacidsofeachchainarehighlightedbyaspecificcolorandcanbedistinguishedfromthoseofotherchains.

Answer

2Howmanymonomers/chainsdoyouseeinthecurrentviewofPFK(2PFK)?• TodistinguishbetweenthedifferentmonomersthatmakeupPFK(2PFK),underStructureclickonQuaternary.

• ClickonChain.

Answer

3DoesthecurrentviewofPFK(2PFK)representtheactiveformofthisenzyme?AnswerYes/Noandexplainyouranswer.

Answer

4Letushighlightaminoacids#195,#196and#224inthepolypeptidechain.• UnderStructure,clickonPrimary.• SelecttheseaminoacidresiduesbysimultaneouslyclickingonControlandApplekey(Mac)/right‐click(PC).• Theaminoacidsyouselectgethighlightedinthecrystalstructure(white).ForabetterviewyoucangotoViewControlsandmovetheUnselectedtransparencysliderto“0”.

• WithintheAtomsboxclickonDrawtovisualizetheatomspresent.Eachatomiscolor‐coded:Carbonisgrey,Nitrogenisblue,OxygenisredandinthisexerciseIronisorange.

• AlternativelymovetheBackboneslidertothelefttovisualizetheatomsthatareinvolvedinbonding/interaction.Youcanzoomintheselectedaminoacidsforabetterview.

a)Whatisthemostlikelytypeofinteractionbetweenaminoacids#196and#224?Yourchoicesare‘hydrogenbond’,‘ionicbond’,‘hydrophobicinteraction’,‘covalentbond’,‘peptidebonds’or‘vanderWaalsforces’.

Answer

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b)Whatisthemostlikelytypeofinteractionbetweenaminoacids#195and#196?Yourchoicesare‘hydrogenbond’,‘ionicbond’,‘hydrophobicinteraction’,‘covalentbond’,‘peptidebonds’or‘vanderWaalsforces’.

Answerc)Drawaminoacids#195and#196andcirclethereactivegroup(s)oratomsthataccountfortheirinteraction.Explainwhyyouselectedthesegroups(atoms).

Answer

d)Arethesethreeaminoacidspartofthesameordifferentpolypeptidechain?• WithinStructure,gotoQuaternaryandclickonChain.• Examinethecolorassociatedwiththeseaminoacidstodetermineiftheyarethesameordifferent.

Answer

e)Highlightaminoacids#241and#261.Whatisthemostlikelytypeofinteractionbetweenthesetwoaminoacids?Yourchoicesare‘hydrogenbond’,‘ionicbond’,‘hydrophobicinteraction’,‘covalentbond’,‘peptidebonds’or‘vanderWaalsforces’.

Answer

5Individualaminoacidsfoldintodifferentlocalsecondarystructures.

a)NamethedifferentsecondarystructuresobservedinPFK(2PFK)?• UnderStructure,clickonSecondary.• ExplorethedifferenttypesofsecondarystructuresfoundinPFK(2PFK)byindividuallyclickingonthedifferentchoicesthatappearwithintheShowRibbonsbox.

Answer

b)PleaselistthenumberofindividualstructuresyouobserveforeachofthesecondarystructuresinPFK(2PFK).FocusonjustoneofthePFKmonomers.

Answer

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c)Acleftisfoundbetweenthetwodomainsthatformtheactivesitewithineachmonomer.Thiscleftisformedbytwosheetswhichareangledtowardseachother.Identifythesheetswithinthestructurethatcontributetotheformationoftheactivesite.ChooseonlyoneofthetwoindependentPFKstructureswhenansweringthisquestion.

Answer

d)WewillnowexamineoneofthesheetsfoundwithinPFK(2PFK):S1C.IsS1Canexampleofaparallelorananti‐parallelsheet?• WithinSecondary,clickonTrackSelection.• WithinSheetSelection,clickon“S1C”.• CloseStructureandclickonViewControls.• MovetheUnselectedtransparencyslidertotheleftuntiltherestoftheproteinfallsintothebackgroundorcompletelydisappears.Zoomintheselectedhelixalsoforabetterview.

• TodeterminethelocationofthesidechainsmovetheSidechaintransparencyslidertotheleft,whilekeepingtheBackbonetransparencyslidertotheright.RepeatthisbymovingtheBackbonetransparencyslidertotheleftwhilekeepingtheSidechaintransparencyslidertotheright.

• ComparetheorientationoftheOxygenatoms(red)withintheC=0groupsofeachaminoacidinonepleatofthesheetwiththelocationofthesameoxygenatomsinthenextpleat.

Answer

6ExplorethetertiarystructureofPFK(2PFK)anditsrelationshipwiththeenvironment.• UnderView,clickonResetmolecule.• ClickonStructureandselectTertiary.• WithinColorbyResidueboxclickeitherindividuallyorincombinationthedifferentoptionsprovidedtoanswerthefollowingquestions.

• ToimprovevisualizationoftheproteincloseStructure.• ClickViewControls.• WithintheAtomsboxclickonDrawandFillSpacetochangetheway2PFKisviewed.

Whyistheobserveddistributionofnon‐polaraminoacidswithinPFK(2PFK)criticalforthelocalizationandfunctionofthisenzymeinthecytosol?

Answer

ProteinStructure‐>FunctionQuestions7WewillnowexplorePFK’scatalyticactivity.TodothiswewillcomparethestructureofPFKindifferentstates.• ClickonViewandchooseResetMolecule.• OpentwonewwindowsofStarBiochem,whilekeepingthe2PFKwindowopen.• ClickontheStartbuttontolaunchtheapplication.

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• UnderFileclickonOpen/Import.• Clickon“1PFK”and“6PFK”ineachwindow.• ClickonOpenforbothwindows.• ComparethethreePFKstructuresbygoingthroughtheStructuremenu.• ClickonPDBTree.ComparetheinformationprovidedinPDBTree,inparticulartheligandsboundtothestructures,withthatprovidedinthe“Background”section.

a)Whichofthesethreestructuresrepresentstheresting(ligand‐unbound)stateofPFK?Explainyouranswer.

Answer

b)Nowcomparethestructureofthetworemainingmoleculesthatyoudidnotselectwhileansweringthequestionabove(7a).WhichofthetwostructuresrepresentstheactivestateandwhichonerepresentstheinactivestateofPFK?Explainyourchoices.

Answer

Keywords:Enzymes,regulatory/rate‐limitingreactions,glycolysis,aerobicrespiration,anaerobicrespiration,catalyticreactions,reversibleandirreversiblereactions,allostericmodulators,allostericactivators,andallostericinhibitors.ThoughtQuestions1DeficiencyinPFKleadstoTarui'sdisease.Thisdisorderischaracterizedbyseverenausea,vomitingandmusclecramps.Patientswiththisgeneticdisorderareadvisednottoexercisevigorously.Explainwhyreducedphysicalactivitycanhelpthesepatients.2ActivityofPFKinfluencestheproductionofATPbothbyaerobicandanaerobicrespiration.Explainwhythisisso.3ATPisrequiredforthereactioncatalyzedbyPFK.However,ATPisalsoaninhibitorofPFK.HowcouldATPperformtwodifferentregulatoryeffectsonthesameenzyme?

Download - Phosphofructokinase (PFK) Exercise - STARstar.mit.edu/.../biochem/exercises/pfk_exercise_ver5.pdf · 2012-08-29 · Ver. 5 ‐ D. Sinha and L. Alemán 1 Phosphofructokinase (PFK)

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