Name________________________
StarBiochem
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Phenylalaninehydroxylase&PKUDisorderExerciseLearningObjectivesInthisexercise,youwilluseStarBiochem,aprotein3Dviewer,toexplore:• thestructureoftheenzymephenylalaninehydroxylase(PAH)• therelationshipbetweenthestructureofamutatedformofPAHandthefunctionofthismutatedproteininpatientswithphenylketonuria(PKU),ageneticallyinheriteddisease
BackgroundPhenylketonuria(PKU)isageneticdisorderthatischaracterizedbytheinabilityofthebodytoutilizetheessentialaminoacid,phenylalanine.PKUoccursinapproximately1in10,000newbornsintheUSand1inevery50individualsintheUSisacarrierofPKU.Thisdisorderisequallyfrequentinmalesandfemales.
“ClassicPKU”iscausedbythecompleteornearlycompletedeficiencyoffunctionalphenylalainine(PAH).Thisenzymeispresentbothinprokaryotesandeukaryotes.MammalianPAHoccursasahomodimerinitsrestingstatebutasahomotetramerinitsfunctionallyactiveform.Eachmonomeriscomposedofthreedomains:anN‐terminusregulatorydomain,acentralcatalyticdomain,andashortC‐terminustetramerizationdomainthatplaysaroleininterlockingthemonomerstomakeafunctionallyactivetetramer.
PAHisabundantintheliver.Innormalhealthyindividuals,thisenzymeconvertsphenylalaninetotyrosine,asshowninthefigurebelow.Theseaminoacidsthenmovefromthelivertothebloodstreamandbecomeavailabletocellsformakingproteins.However,inPKUpatientswholackfunctionalPAH,phenylalanineaccumulatesinthebloodandtissues,whereitisconvertedintotoxicproducts.Thesetoxicproductsresultinseverementalretardationduringdevelopment,aswellasseizuresandbehavioraldisorders.
GettingstartedwithStarBiochem• TogettoStarBiochem,pleasenavigateto:http://web.mit.edu/star/biochem.• ClickontheStartbuttontolaunchtheapplication.• ClickTrustwhenapromptappearsaskingifyoutrustthecertificate.• InthetopmenuunderFileclickonOpen/Import.• Select“2PAH”fromtheBundledSamplesfolderandclickOpen.
YouarenowviewingthestructureofPAH(2PAH),witheachbondintheproteindrawnasaline.
+ + PheylphyruvatePhenylacetatePhenyllactatePhenylethylamine
InPKU
Name________________________
StarBiochem
Ver.7‐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
TakeamomenttolookatthestructureofPAH(2PAH)fromvariousanglesinthis"bondsonly"view.Beforeproceedingtoanswerthequestions,youcanreviewthebasicstructuresandtermsonthenextpagewhichyoucanrefertoduringthisexercise.
Name________________________
StarBiochem
Ver.7‐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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ProteinStructureQuestions‐Level11HowmanyaminoacidsarelikelypresentinthePAHstructure:2PAH?• ClickonStructure.• ClickonPrimarywhichshowstheaminoacidsthataresequentiallyjoinedthroughpeptidebondstomaketheamino/polypeptidechain.Theaminoacidsofeachchainarehighlightedbyaspecificcolorandcanbedistinguishedfromthoseofotherchains.
Answer
2Howmanymonomers/chainsdoyouseeinthecurrentviewofPAH(2PAH)?• Todistinguishbetweenthedifferentmonomersthatmakeup2PAH,underStructureclickonQuaternary.• ClickonChain.
Answer
3Glanceattheprimarysequenceofeachmonomer/proteinchain.Aretheprotein/polypeptidechainswithinPAH(2PAH)likelytobeidenticalordifferent?AnswerYes/Noandprovideabriefexplanationforthechoicethatyoumade.
Answer
4DoesthecurrentviewofPAH(2PAH)representtheactiveformofthisenzyme?AnswerYes/Noandexplainyouranswer.
Answer
ProteinStructureQuestions‐Level25Individualaminoacidsinamonomerfoldintodifferentlocalsecondarystructures.
a)WhatarethedifferentsecondarystructuresthatyouobserveinPAH(2PAH)?• UnderStructureclickonSecondary.• ExplorethedifferenttypesofsecondarystructuresfoundinPAH(2PAH)byindividuallyclickingonthedifferentchoicesthatappearundertheShowRibbonsselection.
Answer
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StarBiochem
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b)HowmanyofeachtypeofsecondarystructurearepresentineachmonomerofPAH(2PAH)?
Answer
6Let'sexploreoneofthesecondarystructuresfoundinPAH(2PAH).• Withinthemainmenu,clickonViewandchooseResetMolecule.• GotoSecondary.• ClickonTrackSelectionandthenwithintheHelixselectionclickon“Helix32”.
a)Inwhichdirectiondotheaminoacidsidechainspoint:towardsorawayfromthehelix?• CloseStructureandclickonViewControls.• MovetheUnselectedtransparencyslidertotheleftuntiltherestoftheproteinfallsintothebackgroundordisappears.
• MovetheSidechaintransparencyslidertothelefttodeterminethelocationofthesidechains.YoucanrepeatthisbymovingtheBackbonetransparencyslidertotheleftwhilekeepingtheSidechaintransparencyslidertotheright.
• ClickonDrawwithintheAtomsboxandzoominthehelixforabetterview.
Answer
b)Howmanyaminoacidsarelikelypresentineachturnofahelix?Hint:Alltheaminoacidsthatmakehelix#32arehighlightedintheprimarystructureof2PAH.
Answer
ProteinStructureQuestions‐Level37ExplorethetertiarystructureofPAH(2PAH)anditsrelationshipwiththeenvironment.• Withinthemainmenu,clickonViewandchooseResetMolecule.• WithinStructure,clickonTertiary.• ClickeitherindividuallyorincombinationonthedifferentoptionsprovidedintheColorbyResidueselectiontoanswerthefollowingquestions.
• ToimproveyourvisualizationoftheproteincloseStructureandclickonViewControl.WithintheatomsboxclickonDrawandFillSpacetochangethewayinwhichPAH(2PAH)isviewed.
a)Wheredoyoufindmostofthenon‐polaraminoacidswithinPAH(2PAH)?Yourchoicesare:‘hiddenwithintheinnerpartoftheprotein’,‘awayfromthesurroundingenvironment’,or‘onthesurfaceoftheprotein/exposedtothesurroundingenvironment’.
Answer
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StarBiochem
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b)WheredoyoufindmostofthepolaraminoacidswithinPAH(2PAH)?Youhavethesamechoicesasstatedinpart(a).
Answer
c)WheredoyoufindmostofthechargedaminoacidswithinPAH(2PAH)?Youhavethesamechoicesasstatedinpart(a).
Answer
d)Whatarethetwoclassesofchargedaminoacidsyouobserve?
Answer
e)IfinsteadofPAH(2PAH)youwereviewingthestructureofatransmembraneprotein,wouldyouranswertoparts(a)through(c)bethesameordifferent?Explain.
Answer
8Nowlet’sexplorecertainaminoacidswithinthestructureofPAH(2PAH)andtheirpotentialrelationshipwithotheraminoacids.Toanswerallpartsofthisquestion,pleasetakeamomenttolookatthechartofessentialaminoacidsprovidedinthisexercise.Pleasenotethesimilaritiesanddifferencesbetweenallaminoacids.• UnderStructureclickonPrimaryandselectanaminoacidresiduebymouseclick.Theaminoacidresiduethatyouselectgetshighlightedinthestructure(white).
• Zoominthehighlightedaminoacidresidue.• Carefullyanalyzeitsside‐chainbycomparingitsstructurewiththatprovidedintheaminoacidchart.• Clicksequentiallyontheneighboringaminoacidswhoseside‐chainmaybeinteractingwiththeside‐chainoftheaminoacidthatyoupreviouslyselected.
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• TohighlightmorethanoneaminoacidatatimeclickonthemwhilesimultaneouslypressingControlandApplekey(Mac)/right‐click(PC).
a)Whattypeofaminoacidwouldyouexpecttobeclosedtoanacidicaminoacid?Circlethemostappropriateoption(s).
Answeri.Basicii.Acidiciii.Eitheriv.Neither
b)Provideanexplanationfortheoptionsyoudidnotselectinpart(a).
Answer
c)GiveanexamplewithinthePAHstructure(2PAH)toexplaintheoption(s)thatyouhaveselectedinpart(a).
Answer
Structure‐>Function‐>DiseaseQuestions9NowletusexplorehowthestructureofPAHspecifiesitsfunction.Forthispurpose,wewilllookatthecrystalstructureoftwoPAHmonomers:“1J8U”and“1TDW”.• OpentwonewwindowsofStarBiochem.• InthetopmenuunderFileclickonOpen/Import.• Clickon“1J8U”inonewindowand“1TDW”intheotherwindow.ClickOpeninbothwindows.
a)WhichofthetwostructureslikelyrepresentsthefunctionallyactiveformofaPAH?Explainyourchoice.Note:forsimplicityyoumayassumethateachofthesemonomerscanbindtophenylalanine.Hint:Comparethemoleculesshowninyellowinthetwoproteinstructures.Refertothereactionthatisprovidedinthebackgroundsection.
Answer
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StarBiochem
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b)IdentifythemoleculesthatareboundtothefunctionallyactiveformofPAH.
Answerc)NowlookatthePAHstructurethatyoudidnotselectabove.ProposehowthisformofPAHmayaccountforthedevelopmentofPKU.
Answer
Keywords:Essentialaminoacids,phenylketonuria,phenylalaninehydroxylase,prokaryotes,eukaryotes,seizures.ThoughtQuestionsAnsweronlyoneofthesethreequestions1WhyarePKUpatientsadvisedbytheirdoctorstoavoiddietsoda?2Properfunctionofthenervoussystemrequiresafinebalancebetweentherelease,synthesis,uptakeanddegradationofdifferentneurotransmitters.Dopamineandnorepinephrinearecommonexamplesofneurotransmitters.ExtrapolatewhyPKUpatientsmaydemonstrateneurologicabnormalitiesrelatedtodopamineandnorepinephrine.3PKUpatientsarealsoknowntohavealowrateofaerobicrespirationbutanormalrateofglycolysis.Explainwhy.