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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

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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.

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

CHEMICALSTRUCTURESOFTHEAMINOACIDSThe20aminoacidsshareacommonbackboneandaredistinguishedbydifferent‘R’groups,highlightedinvariouscolorsbelow.

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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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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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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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b)IdentifythemoleculesthatareboundtothefunctionallyactiveformofPAH.

Answerc)NowlookatthePAHstructurethatyoudidnotselectabove.ProposehowthisformofPAHmayaccountforthedevelopmentofPKU.

Answer

Keywords:Essentialaminoacids,phenylketonuria,phenylalaninehydroxylase,prokaryotes,eukaryotes,seizures.ThoughtQuestionsAnsweronlyoneofthesethreequestions1WhyarePKUpatientsadvisedbytheirdoctorstoavoiddietsoda?2Properfunctionofthenervoussystemrequiresafinebalancebetweentherelease,synthesis,uptakeanddegradationofdifferentneurotransmitters.Dopamineandnorepinephrinearecommonexamplesofneurotransmitters.ExtrapolatewhyPKUpatientsmaydemonstrateneurologicabnormalitiesrelatedtodopamineandnorepinephrine.3PKUpatientsarealsoknowntohavealowrateofaerobicrespirationbutanormalrateofglycolysis.Explainwhy.