Enzymology at the Membrane Interface: Interfacial Enzymology and Protein-Membrane Binding

409 Pages·2017·15.968 MB· 430 \409

by  Michael H. Gelb (Eds.)

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
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CONTRIBUTORS
S.Amara
CNRS,AixMarseilleUniversit(cid:1)e,EnzymologieInterfacialeetdePhysiologiedelaLipolyse,
Marseille,France
K.Baek
KyungHeeUniversity,Yongin,SouthKorea
S.E.Barbour
UniversityofGeorgiaatAthens,Athens,GA,UnitedStates
S.Bezzine
UniversityofSfax,LaboratoiredeBiochimieetdeG(cid:1)enieEnzymatiquedesLipases,Sfax,
Tunisia
A.B(cid:1)enarouche
CNRS,AixMarseilleUniversit(cid:1)e,EnzymologieInterfacialeetdePhysiologiedelaLipolyse,
Marseille,France
C.Bourlieu
INRACIRAD,UMR1208IATE,Montpellier,France
V.Brglez
InstitutdePharmacologieMol(cid:1)eculaireetCellulaire,Universit(cid:1)edeNiceSophiaAntipolisand
CentreNationaldelaRechercheScientifique(CNRS),Valbonne,France
J.E.Burke
UniversityofVictoria,Victoria,BC,Canada
C.Cambillau
Aix-MarseilleUniversit(cid:1)e,CNRS,ArchitectureetFonctiondesMacromol(cid:1)ecules
Biologiques,Marseille,France
F.Carrie`re
CNRS,AixMarseilleUniversit(cid:1)e,EnzymologieInterfacialeetdePhysiologiedelaLipolyse,
Marseille,France
J.F.Cavalier
CNRS,AixMarseilleUniversit(cid:1)e,EnzymologieInterfacialeetdePhysiologiedelaLipolyse,
Marseille,France
W.Cho
UniversityofIllinoisatChicago,Chicago,IL,UnitedStates;KyungHeeUniversity,
Yongin,SouthKorea
G.L.Dornan
UniversityofVictoria,Victoria,BC,Canada
G.Du
UniversityofTexasHealthScienceCenteratHouston,Houston,TX,UnitedStates
xi
xii Contributors
S.Feng
EuropeanMolecularBiologyLaboratory(EMBL),Heidelberg,Germany;NCCRChemical
Biology,UniversityofGeneva,Geneva,Switzerland
M.A.Frohman
CenterforDevelopmentalGenetics,StonyBrookUniversitySchoolofMedicine,Stony
Brook,NY,UnitedStates
M.H.Gelb
UniversityofWashington,Seattle,WA,UnitedStates
F.Ghomashchi
UniversityofWashington,Seattle,WA,UnitedStates
J.Gomez-Cambronero
WrightStateUniversity,BoonshoftSchoolofMedicine,Dayton,OH,UnitedStates
E.E.Ha
CenterforDevelopmentalGenetics,StonyBrookUniversitySchoolofMedicine,Stony
Brook,NY,UnitedStates
K.M.Henkels
WrightStateUniversity,BoonshoftSchoolofMedicine,Dayton,OH,UnitedStates
Y.Hu
UniversityofIllinoisatChicago,Chicago,IL,UnitedStates
L.Jeammet
InstitutdePharmacologieMol(cid:1)eculaireetCellulaire,Universit(cid:1)edeNiceSophiaAntipolisand
CentreNationaldelaRechercheScientifique(CNRS),Valbonne,France
M.L.Jenkins
UniversityofVictoria,Victoria,BC,Canada
H.Kim
UniversityofIllinoisatChicago,Chicago,IL,UnitedStates
G.Lambeau
InstitutdePharmacologieMol(cid:1)eculaireetCellulaire,Universit(cid:1)edeNiceSophiaAntipolisand
CentreNationaldelaRechercheScientifique(CNRS),Valbonne,France
C.C.Leslie
NationalJewishHealth,Denver;UniversityofColoradoDenver,Aurora,CO,UnitedStates
S.-L.Liu
UniversityofIllinoisatChicago,Chicago,IL,UnitedStates
S.Longhi
Aix-MarseilleUniversit(cid:1)e,CNRS,ArchitectureetFonctiondesMacromol(cid:1)ecules
Biologiques,Marseille,France
E.Mateos-Diaz
CNRS,AixMarseilleUniversit(cid:1)e,EnzymologieInterfacialeetdePhysiologiedelaLipolyse,
Marseille,France
Contributors xiii
Y.Miki
LipidMetabolismProject,TokyoMetropolitanInstituteofMedicalScience,Tokyo,Japan
A.J.Morris
TheGillHeartInstitute,CollegeofMedicine,LexingtonVeteransAffairsMedicalCenter,
UniversityofKentucky,Lexington,KY,UnitedStates
M.Murakami
LipidMetabolismProject,TokyoMetropolitanInstituteofMedicalScience;
AMED-CREST,JapanAgencyforMedicalResearchandDevelopment,Tokyo,Japan
R.Murase
LipidMetabolismProject,TokyoMetropolitanInstituteofMedicalScience,Tokyo,Japan
C.Payr(cid:1)e
InstitutdePharmacologieMol(cid:1)eculaireetCellulaire,Universit(cid:1)edeNiceSophiaAntipolisand
CentreNationaldelaRechercheScientifique(CNRS),Valbonne,France
F.Philip
CenterforDevelopmentalGenetics,StonyBrookUniversitySchoolofMedicine,Stony
Brook,NY,UnitedStates
V.Point
CNRS,AixMarseilleUniversit(cid:1)e,EnzymologieInterfacialeetdePhysiologiedelaLipolyse,
Marseille,France
D.M.Raben
JohnsHopkinsUniversity,Baltimore,MD,UnitedStates
S.Ramanadham
ComprehensiveDiabetesCenter,UniversityofAlabamaatBirmingham,Birmingham,AL,
UnitedStates
A.Roussel
Aix-MarseilleUniversit(cid:1)e,CNRS,ArchitectureetFonctiondesMacromol(cid:1)ecules
Biologiques,Marseille,France
L.Sams
CNRS,AixMarseilleUniversit(cid:1)e,EnzymologieInterfacialeetdePhysiologiedelaLipolyse,
Marseille,France
H.Sato
LipidMetabolismProject,TokyoMetropolitanInstituteofMedicalScience,Tokyo,Japan
M.Schifferer
EuropeanMolecularBiologyLaboratory(EMBL),Heidelberg,Germany
C.Schultz
EuropeanMolecularBiologyLaboratory(EMBL),Heidelberg,Germany
M.A.Seeliger
CenterforDevelopmentalGenetics,StonyBrookUniversitySchoolofMedicine,Stony
Brook,NY,UnitedStates
xiv Contributors
R.Sheng
UniversityofIllinoisatChicago,Chicago,IL,UnitedStates
F.Stein
EuropeanMolecularBiologyLaboratory(EMBL),Heidelberg,Germany
Y.Taketomi
LipidMetabolismProject,TokyoMetropolitanInstituteofMedicalScience,Tokyo,Japan
S.A.Tatulian
CollegeofSciences,UniversityofCentralFlorida,Orlando,FL,UnitedStates
L.W.R.Tay
UniversityofTexasHealthScienceCenteratHouston,Houston,TX,UnitedStates
B.Tu-Sekine
JohnsHopkinsUniversity,Baltimore,MD,UnitedStates
O.Vadas
PharmaceuticalSciencesSection,UniversityofGeneva,Geneva,Switzerland
V.Vi(cid:1)e
Universit(cid:1)eRennes1,InstitutdePhysiquedeRennes,UMRCNRS6251,Rennescedex,
France
Z.Wang
UniversityofTexasHealthScienceCenteratHouston,Houston,TX,UnitedStates
K.Yamamoto
LipidMetabolismProject,TokyoMetropolitanInstituteofMedicalScience;PRIME,Japan
AgencyforMedicalResearchandDevelopment,Tokyo;FacultyofBioscienceand
Bioindustry,TokushimaUniversity,Tokushima,Japan
Y.Yoon
KonkukUniversity,Seoul,SouthKorea
B.Yun
NationalJewishHealth,Denver,CO,UnitedStates
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