phosphofructokinase (pfk) exercise -...
TRANSCRIPT
Name________________________
StarBiochem
Ver.5‐D.SinhaandL.Alemán
1
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
Name________________________
StarBiochem
Ver.5‐D.SinhaandL.Alemán
2
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.
Name________________________
StarBiochem
Ver.5‐D.SinhaandL.Alemán
3
PROTEINSTRUCTUREBASICSEachproteinhasthefollowingthreelevelsofproteinstructure:PrimarystructureListstheaminoacidsthatmakeupaprotein’ssequence,butdoesnotdescribeitsshape.SecondarystructureDescribesregionsoflocalfoldingthatformaspecificshape,likeahelix,asheet,oracoil.TertiarystructureDescribestheentirefoldedshapeofawholeproteinchain.Inaddition,someproteinsinteractwiththemselvesorwithotherproteinstoformlargerproteinstructures.HowtheseproteinsinteractandfoldtoformalargerproteincomplexistermedQuaternarystructure.
CHEMICALSTRUCTURESOFTHEAMINOACIDSThe20aminoacidsshareacommonbackboneandaredistinguishedbydifferent‘R’groups,highlightedinvariouscolorsbelow.
Name________________________
StarBiochem
Ver.5‐D.SinhaandL.Alemán
4
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
Name________________________
StarBiochem
Ver.5‐D.SinhaandL.Alemán
5
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
Name________________________
StarBiochem
Ver.5‐D.SinhaandL.Alemán
6
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.
Name________________________
StarBiochem
Ver.5‐D.SinhaandL.Alemán
7
• 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?