Practical Approaches to Biological Inorganic Chemistry Edited by Robert R. Crichton Batiment Lavoisier Universite´ Catholique de Louvain Louvain-la-Neuve,Belgium Ricardo O. Louro ITQB, UniversidadeNova deLisboa Oeiras,Portugal AMSTERDAM(cid:129)WALTHAM(cid:129)HEIDELBERG(cid:129)LONDON(cid:129)NEWYORK(cid:129)OXFORD PARIS(cid:129)SANDIEGO(cid:129)SANFRANCISCO(cid:129)SYDNEY(cid:129)TOKYO Elsevier Radarweg29,POBox211,1000AEAmsterdam,TheNetherlands TheBoulevard,LangfordLane,Kidlington,OxfordOX51GB,UK Copyright(cid:1)2013ElsevierB.V.Allrightsreserved. Nopartofthispublicationmaybereproduced,storedinaretrievalsystemortransmittedinanyformorbyanymeans electronic,mechanical,photocopying,recordingorotherwisewithoutthepriorwrittenpermissionofthepublisher PermissionsmaybesoughtdirectlyfromElsevier’sScience&TechnologyRightsDepartmentinOxford,UK:phone(+44)(0) 1865843830;fax(+44)(0)1865853333;email:permissions@elsevier.com.Alternativelyyoucansubmityourrequestonline byvisitingtheElsevierwebsiteathttp://elsevier.com/locate/permissions,andselectingObtainingpermissiontouseElsevier material Notice Noresponsibilityisassumedbythepublisherforanyinjuryand/ordamagetopersonsorpropertyasamatterofproducts liability,negligenceorotherwise,orfromanyuseoroperationofanymethods,products,instructionsorideascontainedinthe materialherein.Becauseofrapidadvancesinthemedicalsciences,inparticular,independentverificationofdiagnosesand drugdosagesshouldbemade BritishLibraryCataloguinginPublicationData AcataloguerecordforthisbookisavailablefromtheBritishLibrary LibraryofCongressCataloging-in-PublicationData AcatalogrecordforthisbookisavailablefromtheLibraryofCongress ISBN:978-0-444-56351-4 ForinformationonallElsevierpublicationsvisit ourwebsiteatwww.store.elsevier.com PrintedandboundinChina 1213 1110987654321 Preface Shroudedinthemistsofscientificantiquity(thingsmovesoquicklythatevenadecadeortwoseemsalongtime), inrealityalittlelessthan30yearsagoetheFederationofEuropeanBiochemicalSocieties,betterknownbyits acronym FEBS, invited the Belgian Biochemical society to organise their annual Congress in Belgium. For the first time in the history of these meetings (since the inaugural Congress, in London in 1964), two half day symposiawereorganisedonthesubjectofmetalloproteins.Attheendofthesecondofthese,agroupofwhatin thosedayswerecalledinorganicbiochemistsmettoenjoyadrinktogetherinthebaroftheSheratonHotel.The outcomewasthattwoofthosepresent,oneofwhomisco-editorofthepresentvolume,togetherwithCeesVeeger wereentrustedwiththetaskoforganisingaFEBSWorkshopCourseonInorganicBiochemistry.Thefirstofthese washeldattheHotelEtapinLouvain-laNeuveattheendofApril,1985.Theoriginsofthisbookcanbetraced back tothe long series ofAdvanced Courses which have followed that pioneering start. At that very first Course, the pattern was established of organising lectures to introduce the subject and to present a theoretical background to the methods which could be used to study metals in biological systems, togetherwithpracticalsessionsinsmallergroups.Thefinallectureswerethendevotedtospecificexamples.Itis interesting,andperhapsnottoosurprising,thatafteranintroductiontoligandfieldtheorybyBobWilliams,and metal coordination in biology by Jan Reedijk, X-ray, EPR, NMR, Mo¨ssbauer and EXAFS spectroscopy of metalloproteins were on the programme. The practicals included NMR, EPR and Mo¨ssbauer as well as Cees Veeger’sfavourite,biochemicalanalysisofFeandSinFeeSproteins.TherewasaneveninglecturebyHelmut Beinert (then on sabbatical in Konstanz) entitled ‘Limitations of Spectroscopic Studies on Metalloproteins and ChemicalAnalysisofMetalsinProteins’.Whilethelecturerswereshuffledaroundfromyeartoyear,FredHagen, AntonioXavier,AlfredTrautwein,andDaveGarnerrepresentedthecornerstoneofthespectroscopicpartofthe courseoverthe early years. Since then, over the period from 1985 until now we have organised some 20 courses, and trained over 800 students,mostofwhomweredoctoralorpost-doctoralstudentswhentheycame onthecourse.Itisasourceof greatprideandsatisfactionthatmanyoftheformerstudentsstillenjoyactiveanddistinguishedcareersinthearea of Biological Inorganic Chemistry, as we now call the subject. Even more rewarding are the number offormer participantswhonowformthestaffofthecourse,notablytheotherco-editor,whohasalsotakenonthemantleof co-organiser of the most recent courses. Indeed, with the exception of Rob Robson, who taught the Molecular Biology lectures and practical for many years, the other authors contributing to this book, Frank Neese, Fred Hagen,EckhardBill,MartinFeiters,ChristopheLegerandMargaridaArcherareallalumniofthe‘Louvain-la- Neuve’course. Our intention in editing this volume is that it can serve as a starting point for any student who wants to study metals in biological systems. The presentations by the authors represent a distillation of what they have taughtoveranumberofyearsintheadvancedcourse.Webeginwithanoverviewoftherolesofmetalionsin biological systems, which we hope will serve as taster for the reader, who will find a much more detailed account in the companion work to this volume (Crichton, 2012). Thereafter, after an introduction to that most erudite of discipline (at least for non-inorganic chemists) ligand field theory, augmented by a good dose of how molecular orbital theory can predict the properties of catalytic metal sites. This leads naturally into a sequence which describes the physicochemical methods which can be used to study metals in biology, concluding with an overview of the application of the powerful methods of modern genetics to metalloproteins. ix x Preface The considerations expressed by that pioneer of analytical precision Helmut Beinert in his 1985 evening lectureinLouvain-la-Neuveareasrelevanttodayastheywerethen.Useasmanytechniquesaspossibletoanalyse your sample e the more information from different approaches you have, the better we will understand your protein.Donotwasteexpensiveandsensitivemethodsonshoddyimpuresamples,andconverselydonotemploy primitivetechnicalmeanstoanalysehighlypurifiedsamples,whichhaverequiredenormousinvestmenttoobtain them.Andaboveallrecognisethatthekeytometalloproteincharacterisationiscollaboration.Donotthinkyou cansimplyphagocytiseatechniquefromthelaboratoryofacolleaguewhoknowsthemethodinsideouteitis muchrichertocollaborate,incorporatinghisorherknow-howintoyourresearch.Andyouwillbethericherforit. Bonnechance,goodluck,boasorteeandwelookforwardtogreetyouononeofthecourseswhichwill,we hope,continueintothefuture.Hopefully,thislittleintroductorytextwillnotonlywhetyourappetite,buthelpyou tofindyourway about the myriad practical methods which can be used to study metals inbiological systems. Robert R. Crichton and Ricardo O. Louro Louvain-la-Neuve, July, 2012 Chapter 1 An Overview of the Roles of Metals in Biological Systems Robert R. Crichton BatimentLavoisier,Universite´CatholiquedeLouvain,Louvain-la-Neuve,Belgium Chapter Outline Introduction:WhichMetalsIonsandWhy? 1 SomePhysicochemicalConsiderationsonAlkaliMetals 3 NaDandKDeFunctionalIonicGradients 3 Mg2DePhosphateMetabolism 5 Ca2DandCellSignalling 6 ZinceLewisAcidandGeneRegulator 10 IronandCoppereDealingwithOxygen 12 NiandCoeEvolutionaryRelics 13 MneWaterSplittingandOxygenGeneration 16 MoandVeNitrogenFixation 18 INTRODUCTION: WHICH METALS IONS AND WHY? Inthecompanionbooktothisone,‘BiologicalInorganicChemistry2ndedition’(Crichton,2011),weexplainin greaterdetailwhylifeasweknowitwouldnotbepossiblewithjusttheelementsfoundinorganicchemistrye namely carbon, oxygen, hydrogen, nitrogen, phosphorus and sulfur. We also need components of inorganic chemistry as well, and in the course of evolution nature has selected a number of metal ions toconstruct living organisms. Some of them, like sodium and potassium, calcium and magnesium, are present at quite large concentrations,constitutingtheso-called‘bulkelements’,whereasothers,likecobalt,copper,ironandzinc,are knownas‘trace elements’,with dietary requirements that are much lower than the bulkelements. Justsixelementseoxygen,carbon,hydrogen,nitrogen,calciumandphosphorusemakeupalmost98.5%of theelementalcompositionofthehumanbodybyweight.Andjust11elementsaccountfor99.9%ofthehuman body (the five others are potassium, sulfur, sodium, magnesium and chlorine). However, between 22 and 30 elementsarerequiredbysome,ifnotall,livingorganisms,andofthesearequiteanumberaremetals.Inaddition to the four metal ions mentioned above, we know that cobalt, copper, iron, manganese, molybdenum, nickel, vanadiumandzincareessentialforhumans,whiletungstenreplacesmolybdenuminsomebacteria.Theessential natureofchromiumfor humans remains enigmatic. Justwhytheseelementsoutoftheentireperiodictable(Figure1.1)havebeenselectedwillbediscussedhere. However,theirselectionwaspresumablybasednotonlyonsuitabilityforthefunctionsthattheyarecalleduponto PracticalApproachestoBiologicalInorganicChemistry,1stEdition.http://dx.doi.org/10.1016/B978-0-444-56351-4.00002-6. Copyright(cid:1)2013ElsevierB.V.Allrightsreserved. 1 2 PracticalApproachestoBiologicalInorganicChemistry FIGURE1.1 Anabbreviatedperiodictableoftheelementsshowingthemetalionsdiscussedinthischapter. playinwhat ispredominantlyanaqueousenvironment,butalsoontheir abundanceandtheir availabilityinthe earth’scrustand itsoceans(which constitutethe major proportion of the earth’s surface). The13metalionsthatwewilldiscussherefallnaturallyintofourgroupsbasedontheirchemicalproperties.In þ þ þ (cid:2) the first, we have the alkali metal ions Na and K . Together with H and Cl , they bind weakly to organic ligands,havehighmobility,andarethereforeideallysuitedforgeneratingionicgradientsacrossmembranesand þ þ formaintainingosmoticbalance.Inmostmammaliancells,mostK isintracellular,andNa extracellular,with þ thisconcentrationdifferentialensuringcellularosmoticbalance,signaltransductionandneurotransmission.Na þ andK fluxesplayacrucialroleinthetransmissionofnervousimpulsesbothwithinthebrainandfromthebrain tootherparts ofthe body. Thesecondgroupismadeupbythealkalineearths,Mg2þandCa2þ.Withintermediatebindingstrengthsto organicligands,theyare,atbestsemi-mobile,andplayimportantstructuralroles.TheroleofMg2þisintimately associated with phosphate, and it is involved in many phosphoryl transfer reactions. Mg-ATP is important in muscle contraction, and also functions in the stabilisation of nucleic acid structures, as well as in the catalytic activity of ribozymes (catalytic RNA molecules). Mg2þ is also found in photosynthetic organisms as the metal þ centreinthelight-absorbingchlorophylls.Ca isacrucialsecondmessenger,signallingkeychangesincellular metabolism, but is also important in muscle activation, in the activation of many proteases, both intra- and extracellular,and as amajor component of arange of bio-minerals, including bone. Zn2þ, which is arguably not a transition element,1 constitutes the third group on its own. It is moderate to strongbinding,isofintermediatemobilityandisoftenfoundplayingastructuralrole,althoughitcanalsofulfil averyimportantfunctionasaLewisacid.Structuralelements,calledzincfingers,playanimportantroleinthe regulationof gene expression. Theothereighttransitionmetalions,Co,Cu,Fe,Mn,Mo,Ni,VandWformthefinalgroup.Theybindtightly to organic ligands and therefore have very low mobility. Since they can exist in various oxidation states, they participate in innumerable redox reactions, and many of them are involved in oxygen chemistry. Fe and Cu are constituentsofalargenumberofproteinsinvolvedinelectrontransferchains.Theyalsoplayanimportantrolein oxygen-binding proteins involved in oxygen activation as well as in oxygen transport and storage. Co, together with another essential transition metal, Ni, is particularly important in the metabolism of small molecules like carbon monoxide, hydrogen and methane. Co is also involved in isomerisation and methyl transfer reactions. A major role of Mn is in the catalytic cluster involved in the photosynthetic oxidation of water to dioxygen in plants,and,fromamuchearlierperiodingeologicaltime,incyanobacteria. MoandWenzymes containapyr- anopterindithiolate cofactor, while nitrogenase, the key enzyme of N fixation contains a molybde- 2 numeironesulfur cofactor, in which V can replace Mo when Mo is deficient. Other V enzymes include 1.IUPACdefinesatransitionmetalas“anelementwhoseatomhasanincompletedsub-shell,orwhichcangiverisetocationswithan incompletedsub-shell.” Chapter j 1 AnOverviewoftheRolesofMetalsinBiologicalSystems 3 haloperoxidases.TodatenoCr-bindingproteinshavebeenfound,addingtothelackofbiochemicalevidencefor a biological roleof the enigmaticCr. SOME PHYSICOCHEMICAL CONSIDERATIONS ON ALKALI METALS Beforeconsidering,inmoredetail,therolesofthealkalimetals,NaþandKþ,andthealkalineearthmetals,Mg2þ andCa2þ,itmaybeusefultoexaminesomeoftheirphysicochemicalproperties(Table1.1).Wecanobserve,for þ þ examplethatNa andK havequitesignificantlydifferentunhydratedionicradii,whereas,thehydratedradiiare much more similar. It therefore comes as no surprise that the pumps and channels which carry them across membranes,andwhichcaneasilydistinguishbetweenthem,aswewillseeshortly,transporttheunhydratedions. Althoughnotindicatedinthetable,itisclearthatNaþisinvariablyhexa-coordinate,whereasKþandCa2þcan þ þ adjusttoaccommodate6,7or8ligands.Asweindicatedabove,bothNa andK arecharacterisedbyveryhigh solvent exchange rates (around 109/s), consistent with their high mobility and their role in generating ionic gradientsacrossmembranes.Incontrast,themobilityofMg2þissomefourordersofmagnitudeslower,consistent with its essentially structural and catalytic. Perhaps surprisingly, Ca2þ has a much higher mobility (3(cid:3)108/s), which explainswhy it isinvolvedin cell signalling via rapid changes on Ca2þfluxes. TheselectivebindingofCa2þbybiologicalligandscomparedtoMg2þcanbeexplainedbythedifferencein theirionicradius,aswepointedoutabove.Also,forthesmallerMg2þion,thecentralfieldofthecationdominates itscoordination sphere, whereasforCa2þ,the secondandpossiblyeventhe third, coordinationspheres havean importantinfluenceresultinginirregularcoordinationgeometry.ThisallowsCa2þ,unlikeMg2þtobindtoalarge numberof centres at once. ThehighchargedensityonMg2þasaconsequenceofitssmallionicradiusensuresthatitisanexcellentLewis acid in reactions notably involving phosphoryl transfers and hydrolysis of phosphoesters. Typically, Mg2þ functionsasaLewisacid,eitherbyactivatingaboundnucleophiletoamorereactiveanionicform(e.g.waterto hydroxide anion), or by stabilising an intermediate. The invariably hexacoordinate Mg2þ often participates in structureswherethemetalisboundtofourorfiveligandsfromtheproteinandaphosphorylatedsubstrate.This leaves one or two coordination positions vacant for occupation by water molecules, which can be positioned in a particulargeometry bythe Mg2þ toparticipate inthe catalytic mechanism ofthe enzyme. NAD AND KD e FUNCTIONAL IONIC GRADIENTS How,wemightask,dothepumpsandchannelsresponsiblefortransportacrossmembranesdistinguishbetween þ þ Na andK ions?Studiesoverthelast50yearsorsoofsyntheticandnaturallyoccurringsmallmoleculeswhich bindionshaveestablishedthebasicrulesofionselectivity.Twomajorfactorsappeartobeofcapitalimportance, TABLE1.1 PropertiesofCommonBiologicalCations Ionic Hydrated Ionic Hydrated Exchange Transport Cation radius(A˚) radius(A˚) volume(A˚3) volume(A˚3) rate(sec(cid:2)1) number Naþ 0.95 2.75 3.6 88.3 8(cid:3)108 7e13 Kþ 1.38 2.32 11.0 52.5 109 4e6 Mg2þ 0.65 4.76 1.2 453 105 12e14 Ca2þ 0.99 2.95 4.1 108 3(cid:3)108 8e12 (FromMaguireandCowan,2002). 4 PracticalApproachestoBiologicalInorganicChemistry namely the molecular composition and the stereochemistry (essentially the size) of the binding site. Synthetic moleculeshavebeencreatedwhichselectivitybindLiþ(radius0.60A˚),Naþ(0.95A˚),Kþ(1.35A˚)andRbþ(radius 1.48A˚)bysimplyadjustingthecavitysizetomatchtheion(Dietrich,1985).Nowthatwehavethecrystalstructures of membrane transport proteins, we can begin to understand how ion selectivity is accomplished (MacKinnon, þ þ 2004; GouaxandMacKinnon, 2005). TheNa -selectivebindingsites inthe Na -dependantleucine transporter þ þ LeuTandtheK -selectivebindingsitesintheK channelhavebeendetermined,providingadirectcomparisonof þ þ þ þ þ þ selectivityforNa andK .TheNa andK ionsarecompletelydehydrated,boththeNa andtheK sitescontain þ þ oxygenligands,butbyfarthemostimportantfactordistinguishingNa andK sitesisthesizeofthecavityformed bythebindingsite,whichagreeswellwiththerulesalreadylearnedfromhost/guestchemistry.Whatdetermines alkalimetalcationselectivity,similartothatobservedinionbindingbysmallmolecules,isthattheproteinselects þ þ foraparticularion,Na orK ,byprovidinganoxygen-linedbindingsiteoftheappropriatecavitysize. þ þ MammaliancellsmaintainahighintracellularK (around140mM)andlowintracellularNa (around12mM) þ þ throughtheactionoftheNa ,K -ATPasepresentintheplasmamembrane.Theoverallreactioncatalysedis: 3Naþ(in) þ2Kþ(out) þATP þH O 53Naþ (out)þ 2Kþ (in) þADP þP 2 i Theextrusionofthreepositivechargesforeverytwowhichenterthecell,resultsinatransmembranepotentialof 50e70mV,whichhasenormousphysiologicalsignificance,controllingcellvolume,allowingneuronsandmuscle cells to be electrically excitable, and driving the active transport of important metabolites such as sugars and aminoacids.Morethanone-thirdofATPconsumptionbyrestingmammaliancellsisusedtomaintainthisintra- cellularNaþ(cid:2)Kþ gradient (in nervecellsthiscan rise toup to70%). This thermodynamically unfavourable exchange is achieved by ATP-mediated phosphorylation of the þ þ Na ,K -ATPase followed by dephosphorylation of the resulting aspartyl phosphate residue, which drives conformationalchangesthatallowionaccesstothebindingsitesofthepumpfromonlyonesideofthemembrane atatime.TheATPaseexistsintwodistinctconformations,E andE ,whichdifferintheircatalyticactivityand 1 2 þ þ þ theirligandspecificity(Figure1.2).TheE form,whichhasahighaffinityforNa ,bindsNa ,andtheE .3Na 1 1 þ formthenreactswithATPtoformthe“high-energy”aspartylphosphateternarycomplexE ~P.3Na .Inrelaxing 1 þ to its “low-energy” conformation E -P, the bound Na is released outside the cell. The E -P, which has a high 2 2 þ þ þ affinityforK ,binds2K ,andtheaspartylphosphategroupishydrolysedtogiveE .2K ,whichthenchanges 2 þ conformation to the E form, releasing its 2K inside the cell. The structures of a number of P-type ATPases, 1 includingtheNaþ-Kþ-ATPaseandtheCa2þ(cid:2)ATPaseoftheSarcoplasmicreticulumhavebeendeterminedand are showninFigure1.3. FIGURE1.2 AmodelfortheactivetransportofNaþandKþbytheNaþ-Kþ-ATPase. Chapter j 1 AnOverviewoftheRolesofMetalsinBiologicalSystems 5 FIGURE 1.3 Overall structures and ion-binding site architectures of two P-type ATPases, rabbit sarcoplasmic reticulum Ca2þ-ATPase (SERCA)andpigNaþ,Kþ-ATPase.TheupperpaneldepictsrabbitSERCA(E1ProteinDataBase[PDB]entry1T5S)andpigNaþ-Kþ-ATPase (E2:Pi,PDBentry3KDP).N-,P-,andA-domainsarecolouredred,blueandyellow,respectively;theb-subunitandg-subunitofNaþ,Kþ- ATPasewheatandcyan.Thelowerpaneldepictstheion-bindingsites,viewedapproximatelyperpendiculartothemembraneplanefromthe extracytoplasmicside,intheE1state.Ionligandingresiduesareshownassticks,transmembranehelicesandcalciumionsinSERCAare indicatedbynumbersandgreyspheres,respectively,andthesitessuperposedastransparentspheresontotheNaþ,Kþ-ATPasemodel.Putative bindingsitesforthethirdsodiumionintheNaþ,Kþ-ATPaseareindicatedasgreyellipses.(FromBublitzetal.,2010.ReproducedCopyright 2010withpermissionfromElsevier). MG2D e PHOSPHATE METABOLISM TheintracellularconcentrationoffreeMg2þisabout5(cid:3)10(cid:2)3M,sothatalthoughMg2þ-bindingtoenzymesis relatively weak (K not more than 105M(cid:2)1) and most Mg2þ-dependent enzymes have adequate local concen- a trationsofMg2þfortheiractivity.Mg2þisthemostabundantdivalentcationinthecytosolofmammaliancells, bindsstronglytoATPandADP,andisthereforeextensivelyinvolvedinintermediarymetabolismandinnucleic acidmetabolism.However,likeZn2þ,itisadifficultmetaliontostudy,sinceitisspectroscopicallysilent,withthe consequence that manyspectroscopic studiesonMg2þ enzymes utilise Mn2þas areplacement metal ion. 6 PracticalApproachestoBiologicalInorganicChemistry OfthefiveenzymesselectedintheEnzymeFunctionInitiative,recentlyestablishedtoaddressthechallenge of assigning reliable functions to enzymes discovered in bacterial genome projects, but for which functions have not yet been attributed (Gerlt et al., 2011), three of them are Mg2þ-dependent. We discuss two of them briefly here. Thehaloalkanoicaciddehalogenasesuperfamily(HADSF)(>32,000nonredundantmembers)catalyseadiverse rangeofreactionsthatinvolvetheMg2þ-dependentformationofacovalentintermediatewithanactivesiteAsp. Despitebeingnamedafteradehalogenase,thevastmajorityareinvolvedinphosphoryltransferreactions(Allenand Dunaway-Mariano,2004,2009).WhileATPasesandphosphatasesarethemostprevalent,thehaloaciddehalogenase (HAD)familycancarryoutmanydifferentmetabolicfunctions,includingmembranetransport,signaltransduction andnucleic-acidrepair.Theirphysiologicalsubstratescoveranextensiverangeofbothsizeandshape,rangingfrom phosphoglycolate, the smallest organophosphate substrate, to phosphoproteins, nucleic acids, phospholipids, phosphorylateddisaccharides,sialicacidsandterpenes. InHADenzymes,Aspmediatescarbon-grouptransfertowater(inthedehalogenases)andphosphoryl-group transfer to a variety of acceptors. Thus, the HAD superfamily is unique in catalysing both phosphoryl-group transfer (top) and carbon-group transfer (bottom) (Figure 1.4a). The roles of the four loops that comprise the catalytic scaffold are shown in Figure 1.4b. The activity ‘switch’ is located on loop 4 of the catalytic scaffold (yellow) which positionsone carboxylate residue tofunctionas ageneral base for the dehalogenases and either two or three carboxylates to bind the Mg2þ cofactor essential for the phosphotransferases. CO represents the backbonecarbonyloxygenofthemoietythatistworesiduesdownstreamfromtheloop1nucleophile(red).The side-chainatthispositionisalsousedasanacid-basecatalystbyphosphataseandphosphomutaseHADmembers. Loop2(green)andloop3(cyan)servetopositionthenucleophileandsubstratephosphorylmoiety.Figure1.4c presents aribbondiagram ofthe fold supporting the catalyticscaffold ofphosphonatase. ThemembersofanotherlargesuperfamilyofMg2þenzymes,theenolasesuperfamily(withmorethan6000 nonredundant members) catalyse diverse reactions, including b-eliminations (cycloisomerisation, dehydration and deamination) and 1,1-proton transfers (epimerisation and racemisation). The three founder members of the family are illustrated by mandelate racemase, muconate lactonising enzyme and enolase (Figure 1.5). They all catalysereactionsinwhichthea-protonofthecarboxylatesubstrateisabstractedbytheenzyme,generatingan enolateanionintermediate.Thisintermediate,whichisstabilisedbycoordinationtotheessentialMg2þionofthe enzyme,isthen directed todifferent products inthe enzyme activesites. CA2D AND CELL SIGNALLING Calciumionsplayamajorroleasstructuralcomponentsofboneandteeth,butarealsocruciallyimportantincell signalling.Topreventtheprecipitationofphosphorylatedorcarboxylatedcalciumcomplexes,manyofwhichare insoluble,thecytosoliclevelsofCa2þinunexcitedcellsmustbekeptextremelylow,muchlowerthanthatinthe extracellularfluidandinintracellularCa2þstores.Thisconcentrationgradientgivescellstheopportunitytouse Ca2þasametabolictriggerethecytosolicCa2þconcentrationcanbeabruptlyincreasedforsignallingpurposes bytransientlyopeningCa2þchannelsintheplasmamembraneorinanintracellularmembrane.Theseincreasesin intracellular free Ca2þ concentration can regulate a wide range of cellular processes, including fertilisation, muscle contraction,secretion, learning andmemory andultimately cell death, both apoptotic and necrotic. Extracellular signals often act by causing a transient rise in cytosolic Ca2þ levels, which, in turn, activates agreatvarietyofenzymesthroughtheactionofCa2þ-bindingproteinslikecalmodulin,aswewilldiscussindetail below: this triggers such diverse processes as glycogen breakdown, glycolysis and muscle contraction. In the phosphoinositide cascade (Figure 1.6), binding of the external signal (often referred to as the agonist2 when it provokes a positive response) to the surface receptor R (step 1) activates phospholipase C, either through a G 2.Manydrugshavebeendevelopedeitherasagonistorantagoniststoreceptor-mediatedsignallingpathways,e.g.b-blockersblocktheaction oftheendogenouscatecholaminesadrenaline(epinephrine)andnoradrenaline(norepinephrine)onb-adrenergicreceptors.