Enzymology at the Membrane Interface: Intramembrane Proteases PDF
Preview Enzymology at the Membrane Interface: Intramembrane Proteases
METHODS IN ENZYMOLOGY Editors-in-Chief ANNA MARIE PYLE Departments of Molecular, Cellular and Developmental Biology and Department of Chemistry Investigator, Howard Hughes Medical Institute Yale University DAVID W. CHRISTIANSON Roy and Diana Vagelos Laboratories Department of Chemistry University of Pennsylvania Philadelphia, PA Founding Editors SIDNEY P. COLOWICK and NATHAN O. KAPLAN AcademicPressisanimprintofElsevier 50HampshireStreet,5thFloor,Cambridge,MA02139,UnitedStates 525BStreet,Suite1800,SanDiego,CA92101–4495,UnitedStates TheBoulevard,LangfordLane,Kidlington,OxfordOX51GB,UnitedKingdom 125LondonWall,London,EC2Y5AS,UnitedKingdom Firstedition2017 Copyright©2017ElsevierInc.Allrightsreserved. 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ISBN:978-0-12-812213-6 ISSN:0076-6879 ForinformationonallAcademicPresspublications visitourwebsiteathttps://www.elsevier.com/ Publisher:ZoeKruze AcquisitionEditor:ZoeKruze EditorialProjectManager:HeleneKabes ProductionProjectManager:MageshKumarMahalingam CoverDesigner:GregHarris TypesetbySPiGlobal,India CONTRIBUTORS A.Abdine IcahnSchoolofMedicineatMountSinai,NewYork,NY,UnitedStates K.Akiyama InstituteforFrontierLifeandMedicalSciences,KyotoUniversity,Kyoto,Japan Y.Akiyama InstituteforFrontierLifeandMedicalSciences,KyotoUniversity,Kyoto,Japan E.Arutyunova FacultyofMedicineandDentistry,MembraneProteinDiseaseResearchGroup,University ofAlberta,Edmonton,AB,Canada R.P.Baker JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD,UnitedStates D.M.Bolduc AnnRomneyCenterforNeurologicDiseases,BrighamandWomen’sHospital,Harvard MedicalSchool,Boston,MA,UnitedStates M.Brown UniversityofMissouri,Columbia,MO,UnitedStates J.Chavez IcahnSchoolofMedicineatMountSinai,NewYork,NY,UnitedStates L.Cha´vez-Guti(cid:1)errez UniversityofLeuven;VIBCenterforBrainandDisease,Leuven,Belgium J.W.Cooley UniversityofMissouri,Columbia,MO,UnitedStates B.Cordier Zentrumfu€rMolekulareBiologiederUniversit€atHeidelberg(ZMBH),DKFZ-ZMBH Allianz,Heidelberg,Germany R.E.Dalbey TheOhioStateUniversity,Columbus,OH,UnitedStates B.DeStrooper UniversityofLeuven;VIBCenterforBrainandDisease,Leuven,Belgium;UCLInstituteof Neurology,London,UnitedKingdom M.Dewilde UniversityofLeuven;VIBCenterforBrainandDisease,Leuven,Belgium € O.D.Ekici TheOhioStateUniversity,Newark,OH,UnitedStates xi xii Contributors A.Fukumori BiomedicalCenter(BMC),MetabolicBiochemistry,Ludwig-Maximilians-University Munich;GermanCenterforNeurodegenerativeDiseases(DZNE),Munich,Germany Y.Hizukuri InstituteforFrontierLifeandMedicalSciences,KyotoUniversity,Kyoto,Japan R.D.JiJi UniversityofMissouri,Columbia,MO,UnitedStates N.Ke NewEnglandBiolabs,Ipswich,MA,UnitedStates B.Kretner BiomedicalCenter(BMC),MetabolicBiochemistry,Ludwig-Maximilians-University Munich;GermanCenterforNeurodegenerativeDiseases(DZNE),Munich,Germany B.Lada UniversityofMissouri,Columbia,MO,UnitedStates M.K.Lemberg Zentrumfu€rMolekulareBiologiederUniversit€atHeidelberg(ZMBH),DKFZ-ZMBH Allianz,Heidelberg,Germany M.J.Lemieux FacultyofMedicineandDentistry,MembraneProteinDiseaseResearchGroup,University ofAlberta,Edmonton,AB,Canada S.Liu WashingtonUniversitySchoolofMedicine,St.Louis,MO,UnitedStates W.Li WashingtonUniversitySchoolofMedicine,St.Louis,MO,UnitedStates R.Panigrahi FacultyofMedicineandDentistry,MembraneProteinDiseaseResearchGroup,University ofAlberta,Edmonton,AB,Canada D.Pei TheOhioStateUniversity,Columbus,OH,UnitedStates D.J.Selkoe AnnRomneyCenterforNeurologicDiseases,BrighamandWomen’sHospital,Harvard MedicalSchool,Boston,MA,UnitedStates Y.Shi MinistryofEducationKeyLaboratoryofProteinScience,Tsinghua-PekingJointCenterfor LifeSciences,BeijingAdvancedInnovationCenterforStructuralBiology,SchoolofLife Sciences,TsinghuaUniversity,Beijing,China D.W.Stafford UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,UnitedStates H.Steiner BiomedicalCenter(BMC),MetabolicBiochemistry,Ludwig-Maximilians-University Munich;GermanCenterforNeurodegenerativeDiseases(DZNE),Munich,Germany Contributors xiii K.Strisovsky InstituteofOrganicChemistryandBiochemistry,AcademyofSciencesoftheCzech Republic,Prague,CzechRepublic J.-K.Tie UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,UnitedStates T.Tomita LaboratoryofNeuropathologyandNeuroscience,GraduateSchoolofPharmaceutical Sciences,TheUniversityofTokyo,Tokyo,Japan J.Trambauer BiomedicalCenter(BMC),MetabolicBiochemistry,Ludwig-Maximilians-University Munich,Munich,Germany I.Ubarretxena-Belandia IcahnSchoolofMedicineatMountSinai,NewYork,NY,UnitedStates S.Urban JohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD,UnitedStates S.Veugelen UniversityofLeuven;VIBCenterforBrainandDisease,Leuven,Belgium M.S.Wolfe AnnRomneyCenterforNeurologicDiseases,BrighamandWomen’sHospital,Harvard MedicalSchool,Boston,MA,UnitedStates G.Yang MinistryofEducationKeyLaboratoryofProteinScience,Tsinghua-PekingJointCenterfor LifeSciences,BeijingAdvancedInnovationCenterforStructuralBiology,SchoolofLife Sciences,TsinghuaUniversity,Beijing,China Y.Yang WashingtonUniversitySchoolofMedicine,St.Louis,MO,UnitedStates R.Zhou MinistryofEducationKeyLaboratoryofProteinScience,Tsinghua-PekingJointCenterfor LifeSciences,BeijingAdvancedInnovationCenterforStructuralBiology,SchoolofLife Sciences,TsinghuaUniversity,Beijing,China PREFACE Interfacialenzymologyisconcernedwithenzymesthatmustaccesstheirsub- stratesfromaboundarybetweentwophases(aninterface),themostcommon type being the lipid–water interface of biological membranes (Berg & Jain, 2002). Interfacial enzymes are distinct from noninterfacial enzymes (Gelb, Jain,Hanel,&Berg,1995).Thelatterincludesenzymesinthewaterlayerthat act on substrates in the water layer. One example is hexokinase, which is a water-solubleenzymethatactsonthehighlywater-solublesubstrateglucose. Noninterfacial also include enzymes in the membrane that act on water- soluble substrates. One example is the phosphodiesterase in the visual trans- ductioncyclethathydrolyzescyclicGMP.Thesenoninterfacialenzymeshave incommonthattheiractivesiteshouldaccesssubstrateinthewaterlayereven thoughoneoftheenzymesisanintegralmembraneprotein.Presumablythe active siteis well exposed tothewater layer evenif the enzyme is bound to membranes.Noninterfacialenzymesaresensitivetotheconcentrationofsub- strateintheaqueousphase(molesofsubstratepervolumeofaqueousphase). Rateequationsdescribingnoninterfacialenzymesarecomposedofratecon- stants andaqueous phaseconcentrationsofinteracting partners (enzyme and substrate). By contrast, interfacial enzymes must access their substrates from theinterface,andtherateequationsthatdescribetheiractiondependonrate constantsandthemolesofsubstratepervolumeofinterface.Amonginterfa- cialenzymes,wealsoincludethosethataccesstheirsubstratesfromthemem- brane core, and in these cases the rate equation contains terms for the concentration of substrate in the membrane core. For enzymes that acton highly water-insoluble substrates,for example, phospholipidswithlongfattyacylchainsortransmembranesegmentsofpro- teins, the concentration of substrate in the aqueous phase is vanishingly small, so much so that the enzyme is forced to access its substrate from the membrane phase. These are the interfacial enzymes. Strictly speaking, itisnotpossibletoconcludethatanenzymeisinterfacialbasedonsolubility argumentsalone.Proofcomesfromtheobservationofprocessivebehavior. Consider the enzyme secreted phospholipase A . This is a water-soluble 2 enzymethatabsorbsontothesurfaceofphospholipidvesiclesandthusexists in a water-soluble state (E) and an interfacial state (E*). It has been exper- imentally demonstrated under some conditions that enzyme prebound to vesiclesofonetypeofphospholipidisnotabletoactonvesiclesofadifferent xv xvi Preface phospholipid added later (under conditions where there is no intervesicle transfer of phospholipids), yet if enzyme is added to a premixture of both vesicles, it acts on both types of phospholipids (Gelb et al., 1995). Enzyme boundtothefirstvesicleisabletoremainboundtothevesicleandcatalyze the hydrolysis of several phospholipids before leaving the interface. If the enzymewasactingonthetraceamountofphospholipidinthewaterphase, this type of processive process would clearly not be possible. These results alsoshowthatbindingofenzymetotheinterface(EtoE*)isnotthesame stepasloadingtheactivesiteoftheenzymewithsubstrate(Michaeliscom- plexformation,E*toE*S).Ifthesestepswerethesame,releaseofproduct from the enzyme’s active site would necessarily result in release of enzyme from the membrane interface to the water layer and processive behavior would not bepossible. Processive behavior of interfacial enzymes has been called“scooting”byMahendraJainandcoworkers,whereasnonprocessive behavior is called “hopping” (Jain & Berg, 1989). An interesting dilemma results with an enzyme that acts on a substrate that has significant solubility in both the membrane and aqueous phases. One example is platelet-activating factor acetylhydrolase that cleaves the ester of a phospholipid containing a short acetyl group instead of a long- chainfattyacylgroup.Theenzymeiswatersolublebutfoundinvivobound tightlytolipoproteinsinblood.Doestheenzymeaccessitssubstrateonlyin theaqueousphase(forexample,themembrane-boundenzymehasitsactive siteexposedmainlytothewaterlayer)ordoestheenzymeaccessitssubstrate onlyinthelipoprotein(forexample,itsactivesiteisnotwellexposedtothe aqueous phase)? The question is nontrivial to answer because the substrate readilypartitionsbetweentheaqueousandmembranephases.Thus,atequi- librium, the concentration of substrate in the membrane phase is equal to the concentration of substrate in the aqueous multiplied by the partition equilibrium constant. Also during equilibrium, variation of the concentra- tionofsubstrateinonephaseleadstoaproportionalvariationofsubstratein the other phase. Any steady-state rate equation that is written in terms of moles of substrate in the aqueous phase divided by the volume of the aqueous phase (conventional concentration) can be rewritten in terms of the moles of substrate in the membrane phase divided by the volume of membranephase(interfacialconcentration)timesaconstant.Thetwoequa- tions are mathematically equivalent, and it is thus impossible to design any steady-state kinetic experiment to determine which equation applies, i.e., whether the enzyme is interfacial or not. The problem has been solved forplatelet-activatingfactoracetylhydrolasebystudyingthekineticsduring Preface xvii the presteady-state phase in which the substrate has not yet equilibrated between membrane and aqueous phases (Min et al., 1999). This study showedthattheenzymeoperatesonplatelet-activatingfactorintheaqueous phase and is thus not an interfacial enzyme. One may wonder whether a membrane-bound signaling receptor that is gated by a ligand that can exist inthemembraneandaqueousphasesinteractswithligandinthemembrane phase (interfacial receptor) or the aqueous phase (noninterfacial receptor). This question has never been answered. For interfacial enzymes that display fast turnover numbers, the catalytic cyclemaybelimitedbytherateofexchangeofsubstratebetweensubstrate aggregatescontainingboundenzyme.Forexample,somesecretedphospho- lipasesA displayaturnovernumberof(cid:1)100s(cid:3)1.Iftheenzymeisboundto 2 asmallunilamellarvesiclecontainingsay500phospholipids,substratewould becomeexhaustedinjustafewseconds.Tocontinue,enzymemustmoveto a new vesicle or there must beintervesicle exchange of phospholipids, and these processes may become rate-limiting for steady-state turnover. In this casethesteady-stateparametersdonotreflectthetruecatalyticpropertiesof theenzymebutratherthoseofsubstrateandenzymeintervesicledynamics. This problem is especially pertinent to phospholipid–detergent mixed micelles that contain only a few phospholipids per particle (Dennis, Cao, Hsu, Magrioti, & Kokotos, 2011). For enzymes such as secreted phospholipase A , there are two compo- 2 nents to their substrate specificity. The first issue is what are the struc- ture–function relationships that determine the affinity of the enzyme for themembranesurface(E toE*).Onceboundto theinterface, thencomes theissueofthestructuralrequirementsoftheinterfacialsubstrateforbinding tothecatalyticsiteofE*togivetheMichaeliscomplexE*S.Forexample, group IIA secreted phospholipase A does not appreciably bind to vesicles 2 that lackanionic phospholipids(for example, vesiclesrichin phosphatidyl- choline), yet once bound to anionic phospholipid vesicles, phosphatidyl- choline that may be present in the same vesicles are good substrates for theenzyme(thus,E*+SÐE*Sisfavorable)(Gelbetal.,1995).Oftensub- stratespecificitystudiesofinterfacialenzymesarecarriedoutinwaysthatdo not allow the deconvolution of these two processes and are thus almost impossible to interpret and to extrapolate from in vitro to in vivo settings. Inhibition of interfacial enzymes is more difficult to study than with noninterfacial enzymes (Gelb et al., 1995). It is possible for inhibitors to actthroughnonspecificmechanisms,forexample,compoundsthatpartition into the membrane interface and change the physical properties of the xviii Preface interface in ways that promote enzyme desorption to the aqueous phase (E*toE).Moreinterestingandusefulinhibitorsarethosethatbindspecifically tothecatalyticsite(orothersite)ontheenzyme(E*+IgivesE*I);theseare analogoustoinhibitorsofnoninterfacialenzymes.Earlystudiesofphospho- lipaseA inhibitorswereplaguedbythecommonoccurrenceofnonspecific, 2 membrane-perturbing inhibitors. For example, annexins were initially characterized as phospholipase A inhibitors, but later studies showed that 2 annexins form a tightly packed array on the membrane surface and simply occludetheinterfacialenzymefrombindingtothevesicletoaccessitssub- strate.Thus,annexins,atleastinvitro,areinhibitorsofvirtuallyallinterfacial enzymesthatmustundergoanEtoE*transitionaspartofitscatalyticcycle. Volume 583 of Methods in Enzymology is focused on interfacial enzymes thatactonlipidsubstrates.Methodsfordetectingandquantifyingthebinding ofproteinsandenzymestomembranesinvitroarecovered inChapters1,9, 10, and 11. Methods for detection of interfacial binding of proteins and enzymes to membranes in living cells are covered in Chapters 2, 4, 6, 13, and 15. Some interfacial enzymes form protein–protein complexes, and this isdescribedinChapters8and14.Studiestoevaluatethesubstratespecificityof interfacialenzymesonnaturalmembersarecoveredinChapter5.Novelspec- troscopic studies for providing structural insight into protein–membrane binding are provided in Chapters 7, 9, 11, and 12. Methods for production of recombinant interfacial enzymes are given in Chapters 3 and 6. Volume584ofMethodsinEnzymologyisfocusedonproteolyticenzymesthat accesstheirtransmembranepeptidesubstratesinthemembranephase(interfa- cial proteases). Biochemical characterization of bacterial and eukaryotic trans- membrane proteases is covered in Chapters 1, 2, 4, 5, 6, 7, 10, and 15. Kinetic studies including substrate specificity and inhibition are included in Chapters2,4,5,6,9,11,12,and15.Studiesthatprobethestructureoftrans- membrane proteases are given in Chapters 3, 6, 8, and 13. Production of recombinanttransmembraneproteasesisthespecificfocusofChapters5and10. Insummary,interfacialenzymesareanimportantsubsetofenzymes.Early studieswerefocusedonmembranesthatactatthelipid–waterinterface.More recentlyanewclassofproteasesthatactontransmembraneproteinsegments have been discoveredin severalorganisms. Specialmethods are requiredfor thecharacterizationofinterfacialenzymesincludingtheirbasicfeaturesofsub- stratespecificityandinhibition.Structuralstudiesarechallengingbecausethe enzyme acts in an environment that is not amenable to conventional tech- niques for determining molecular structure. MICHAEL H. GELB University of Washington, Seattle, WA, United States Preface xix REFERENCES Berg,O.G.,&Jain,M.K.(2002).Interfacialenzymekinetics.WestSussex,England:Wiley. Dennis,E.A.,Cao,J.,Hsu,Y.-H.,Magrioti,V.,&Kokotos,G.(2011).PhospholipaseA2 enzymes: Physical structure, biological function, disease implication, chemical inhibi- tion, and therapeutic intervention. Chemical Reviews, 111, 6130–6185. http://dx.doi. org/10.1021/cr200085w. Gelb,M.H.,Jain,M.K.,Hanel,A.M.,&Berg,O.(1995).Theinterfacialenzymologyof glycerolipidlipases:LessonsfromsecretedphospholipasesA2.AnnualReviewofBiochem- istry,64,653–688. Jain,M.K.,&Berg,O.(1989).ThekineticsofinterfacialcatalysisbyphospholipaseA2and regulationofinterfacialactivation:Hoppingversusscooting.BiochimicaetBiophysicaActa, 1002,127–156. Min,J.H.,Jain,M.J.,Wilder,C.,Paul,L.,Apitz-Castro,R.,Aspleaf,D.C.,etal.(1999). Membrane-bound plasmaplateletactivating factoracetylhydrolaseactsonsubstratein theaqueousphase.Biochemistry,38,12935–12942.
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