Name________________________ StarBiochem Ver. 5 ‐ D. Sinha and L. Alemán 1 Phosphofructokinase (PFK) Exercise Learning Objectives In this exercise, you will use StarBiochem, a protein 3D viewer, to explore the structure and function of the enzyme phosphofructokinase (PFK). Background Living organisms, both unicellular and multicellular, require energy to live and function. Energy is stored and provided in the form of adenosine triphosphate (ATP). ATP is produced by multiple cellular processes including glycolysis, as well as aerobic and anaerobic respiration. PFK is the most important regulatory enzyme of glycolysis. It irreversibly catalyzes the rate‐limiting step of glycolysis: the conversion of fructose ‐6‐phosphate and ATP to fructose‐1, 6‐diphosphate and ADP. Fructose 6‐phosphate fructose 1,6‐biphosphate PFK exists as a homotetramer in bacteria and mammals. Each of the four monomers of PFK is composed of two similar domains. One domain has an ATP binding pocket. The other domain contains the substrate‐ binding site and the allosteric site. An allosteric site is a regulatory binding site that affects enzyme activity and is distinct from the substrate‐binding site of an enzyme. Various activators and inhibitors regulate PFK. Examples of PFK activators are ADP, AMP and fructose 2, 6 diphosphate. Examples of PFK inhibitors are ATP, phosphoglycerate (PGA) and citrate. ADP fructose 2, 6 diphosphate PGA ATP Getting started with StarBiochem • To being using StarBiochem, please navigate to: http://web.mit.edu/star/biochem. • Click on the Start button to launch the application. • Click Trust when a prompt appears asking if you trust the certificate. • Under File, click on Open/Import and select “2PFK” and click Open. You are now viewing the structure of PFK (2PFK) with each bond in the protein drawn as a line (“bonds only” view). You will see two independent PFK structures within the 2PFK file. This is an artifact of how the structure was determined. Please focus on one of the two structures while answering the questions in this exercise. ATP H + + ADP
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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
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StarBiochem
Ver.5‐D.SinhaandL.Alemán
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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.
Name________________________
StarBiochem
Ver.5‐D.SinhaandL.Alemán
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PROTEINSTRUCTUREBASICSEachproteinhasthefollowingthreelevelsofproteinstructure:PrimarystructureListstheaminoacidsthatmakeupaprotein’ssequence,butdoesnotdescribeitsshape.SecondarystructureDescribesregionsoflocalfoldingthatformaspecificshape,likeahelix,asheet,oracoil.TertiarystructureDescribestheentirefoldedshapeofawholeproteinchain.Inaddition,someproteinsinteractwiththemselvesorwithotherproteinstoformlargerproteinstructures.HowtheseproteinsinteractandfoldtoformalargerproteincomplexistermedQuaternarystructure.
CHEMICALSTRUCTURESOFTHEAMINOACIDSThe20aminoacidsshareacommonbackboneandaredistinguishedbydifferent‘R’groups,highlightedinvariouscolorsbelow.
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StarBiochem
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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
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
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StarBiochem
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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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StarBiochem
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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?
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