Energy, Enzymes & Membranes First Term All

Energy, Enzymes & Membranes First Term All -...

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Unformatted text preview: Energy&Thermodynamics October  ­2 8  ­0 9 10:30AM NOTE:Identicaltothetextbookchap.Sincehewrotei t. Energy:abilitytodowork Kinetic=Eass.w/motion Potential=storedEe sp.chemicalpotentialE. Eassociatedduetopositionorchemicalstructure(bonding) Glucosehaspot.Eduetobonds&thewayatomsarearranged Whereyouarei nthegravitationalfielddeterminestheamt.youhave Thermodynamics:studyofhowEistransformed MustdefineWHATwearel ookingat Systemvs.surroundings Outsidesystem=surroundings Isolatedsystem=neitherENORmassmovethrough Closedsystem=onlyEcanbee xchanged Ex.Earth ­ veryl ittlemattere xchanged,juste nergy(sunlight,e tc.) Opensystem=bothE&masse xchanged Ex.humans LawsofThermodynamics: potential kinetic 1stl aw TotalEi sconstant CantransformEtodiff.forms V ibration,heat,e tc. 2ndl aw Etransformationsarenever100%e fficient AlwayssomeEi sl osttoe nvironmenti nformofheat,friction,e tc. i .e.RandomMolecularMotion Increasei ndisorder,randomness=ENTROPY Entropyofasystem&surroundingsalwaysGOESUP(increases) DuringphasechangesEntropyi ncreases Solid ­ ­>l iquid ­ ­>gas Suddenchangewhenphasechangeoccurs. Ex.Physicaldisintegrationofobjects(ex.cars) DoesnotrequireEi nputtooccur...justproceeds WAYOFQUANTIFYINGPOT.EOFAMOLECULE Enthalpy(H)=e nergy(heatcontent)i nasystem  ­ H=e xothermic=TENDtobespontaneous Productshavel esspot.Ethanreactants + H=endothermic=notspontaneous HAVEtoaccountforbothe nthalpy&e ntropy ­ ­>GIBBS! Ex.Glucosewillspontaneouslybreakdown Hasahighpot.Ethereforewantstobreakdowni nCO 2 Entropy(S)i ncreases Energy, Enzymes & Membranes Page 1 Amt.ofEavailabletodowork=FreeE Won'thavetoplugi n#sBUTneedtounderstandwhati tmeans.... HighFreeE=l essstability Enthalpy=ONEcomponentofwhetherareactioni se xergonicORe ndergonic =pot.Eofamolecule ­ changei nchaos(temp.) SPONTANEOUS Increasedstability G<0 EXERGONIC NON ­SPONTANEOUS Decreasedstability G>0 ENDERGONIC NeedanEsource tooccur. H=  ­82kJ/mol G=  ­218kJ/mol Spontaneousrxn  ­ T S=  ­136kJ/mol H=95kJ/mol - Phasechange( entropyi ncreases) - Spontaneousrxns      ­T S= ­320kJ/mol G= ­225kJ/mol Fermentationofglucose Largechangei ne nthalpy Pot.Eofproducts<reactants Exothermicrxn Entropyi ncreases Sold ­ ­>l iquid+gas Result:e xergonicrxn Meltingofi ce Enthalpyi ncreases Bringingi nheat Endothermicrxn Entropyi ncreases Hugei ncreasei ndisorderduetophasechange Entropydrivenrxn MorefreeE,l essstable,greaterworkcapability Glucose:l otsof pot.E Diffusion:changein entropy(entropy driven) LessFreeEnergy,morestable,l essworkcapacity Ane xergonicrxn, G<0 Ex.Cellular resp. Ane ndergonicrxn. G>0 Ei nputreq'd Ex.Photosynthesis &proteinsynthesis CatabolicPathway OverallG= ­7.5kcal/mol Notnecessarilyall individualGsare ­ ve AnabolicPathway OverallG=+4.7kcal/mol Energy, Enzymes & Membranes Page 2 G<0 G=0 Closedsystem AllclosedsystemsreachEQUILIBRIUM G=0 Max.stability G<0 Opensystems:never@e quilibrium Freemvt.ofmatter Iam...ani slandofl owe ntropy Livingthingsarehighlyordered&puttogether Lowe ntropysystems NOTclosedBUTopen Bringi nE.fromsurroundings Usedtomakemoleculesw/highfreeE(tomake&maintainorder) Energy. HEAT Entropyofsurroundingsi ncreases Eattomaintainl owe ntropy ­ donebybringinghighfreeEmolecules Proteinsarebreakingdownconstantlyduetoe ntropy Ifdon'te ati .e.e ache quilibriumthenG=0whichi sBAD...becauseyou'redead. Energy, Enzymes & Membranes Page 3 Enzymes November  ­0 1  ­0 9 11:46PM ALLExergonicrxns ATP G>0(Endergonic) NeedEtooccur!(notspontaneous) ComesfromhydrolysisofATP i.e.hydrolysisof ATP uncatalysed Spontaneitydoesnotcorrelatetotherateofthe reaction ONLYmeansthati tdoesn'tnotrequiree xternalE input Useofe nzymes=catalyststherebyi ncreasingtherate ofreaction Enzymesdon'tprovideE(NOTanEsource),they justspeedupthereaction Doesnotmakerxne xergonici fi t'se ndergonic NeedATPi fe ndergonic HydrolysisofATPl inkedtoe ndergonic processes 1012 ­1020 G<0(EXERGONIC) Spontaneous DoesnotrequireE Catalyzedbye nzymes(i.e. proteins) Transitionstate EA G= ­ve  ­ve G FreeEofproducts<reactants ForreactiontoproceedneedEtocomei ntosystem Emaycomef romsystemi tself Bouncingaround,hittinge achother,e tc. Transitionstate=wherebondsstarttobreak EA =Ereq'dtoreachtransitionstate HowmuchFreeEreq'd EA =akinetic barrier RatemaybeslowbecauseEA maybeveryhigh Propane=classiccase Thermodynamicallyunstable Spontaneouslybreakdown Exergonicprocess BUTi tcansitf or100sofyearssincekineticallystablesinceEA is veryhigh SparkprovidesEA req'df orreactiontooccur&barrieri s overcome Biologicalcatalyst Ratei sproportionaltothe#ofactivatedmolecules NotchangingfreeEofproductsorreactantsbutthePATHthattherxntakes Don'tneedasmuchEtogettotransitionstate Lowerthebarrierallowsmoremoleculestoreachtransitionstate Adropof1.4kcal/molgetsyou10 ­foldi ncreasei nrate Onlyal ittlebiti sreq'dtohaveahugei ncreasei ntherateofrxn EA Whyaree nzymesreq'd? Biologyi scold.Livingorg.needl owtemp(cannotjustheati tup).Proteins denature&membranesfallaparti ftemp.getstoohigh. Enablesyoutohavehighratesofreaction@temp.thatareconduciveto life.(@achemicallyl owtemp.)   G Tertiary&quaternarystructures Activesite ­ small,3 ­ Dclef/crevice,precision Not"lock&key"(staticmodel)butactuallyi nducedfit(flexible) Smallsubstrate molecule Proteinfoldingi swhatmakesthee nzymefunctional  ­ mustacquirethecorrectconformation Activesite Smalli ncomparisontosizeofthee nzyme Wherecatalysisoccurs V erypreciseforsubstrate Onlyapparentwhenmoleculefoldsi nvivoi ntonormalconfig. Cannote asilybeseenl ooking@theprimarysequenceofthee nzyme Enzymei snotastaticmoleculebutflexible Enzymewrapsaroundsubstrate Activesiteonlyreadilyapparentwhensubstratei nside Changesshapetobindtosubstrate Activesite Energy, Enzymes & Membranes Page 4 Preciseorientationof2substrates.Brings2 substratemoleculestogether.Enzymeforces themi ntothecorrectorientation. Catalyticsite(i.e.e nzyme)mimicsthetransitionstate. ALL3=conditionsyoufind@thetransitionstate Putsthemoleculesi ntheorientationthattheyneedto bei n@thetransitionstate. W/e nzymemanymoremoleculescangetthisconfig. Greater#ofmoleculesreachingthetransitionstate Chargei nteractions.Activesite&substrateare chargedcausinge lectrostatici nteractionsbtwn them&thereforetheyarebroughti ntoright alignment. Conformationalstrain.Substrateneedstobe strainedi nsomeway.Activesiteprovidestheright placethereforesubstratei sattractedtoi t. Turnoverrate=molecules/sec. (cycles/sec) DNApolymerase ­ 15 Substratebinding molecules/sec. Slow Carbonicanhydrase ­ 200,000 molecules/sec. V ERYfast.. Productsreleased catalysis ONEXAM!!!!!!!!!!!!!!!!!!!!!!!!! V max 1/2V max Km Enzymeconc.i sconstant. Howmuchproducti smadei nunittime(i.e.rateofproductcreation)=velocity Producte asytodetect&determinequantitativelysincemaychangecolour,e tc. Productbeingformedasameasureofthesubstrateconc. [S]=substrate. As[S]i ncreasestherate@whichtheproductappearsi ncreases Speedi sproportionalto[S]sinceamt=constant Itfinallysaturates/plateaus SATURATIONKINETICS e nzyme=l imitingfactor Canonlyturnoversubstrate@acertainrate. Saturatessincee nzymecan'tprocesssubstrateanyfaster. S=ine xcess&l imiting=turnovertimeofe nzyme Km =affinitybtwne nzyme&substrate(substrateconc.@1/2V max) Measureofi nteractionbtwne nzyme&substrate Ifhighvelocityreached@l ow[S](i.e.steepcurve)thenK m =HIGHi .e.HIGHaffinity Ifhighvelocityreached@high[S]thenK m =LOWi.e.LOWaffinity Yi sn'ti ttheopp?Wouldn'tKmbel oweraccordingtothegraphi fcurvereached1/2Vmax faster? Catalysisi si nhibitedbyothercompounds Inhibitorcompetesforactivesitesincei tl ooksl ikethesubstrate Sameshape&propertiesassubstrate Attacksactivesite&screwsupe nzyme Bindstoactivesite Overcomebyhighsubstrateconc. If[S]=[I]then50%chancefori nhibitortogeti n Thereforei f[S]>>[I]theni nhibitioncanbeovercome @high[S]therei snodiff.btwnhavingornothavingani nhibitorpresent It'sasi fthei nhibitori snottheresincenochance. @l ow[S]theni thasane ffectonthevelocity. Inhibitorgetsi ntheway V max =same Km=l argeri npresenceofi nhibitor Showsuthati nhibitori spresent Needmoresubstratetoget1/2V max Substrate  Inhibitor [S]=[I] control High[S] +competitor [S]>[I] Km Km Energy, Enzymes & Membranes Page 5 SLIDENOTREQ'D. cellwall.Thiscausesthedaughtercellstobesusceptibletoe xploding,e tc.Thereforeprevents normalbacterialgrowth. Req'd. CELLWALL. Aminoacids Cellwall=peptidoglycan Carbohydrate/proteinmoleculethatmakesupcellwall Transpeptidase(enzyme) Makesapeptidebondwithinpeptidoglycan Aminoacidsi ncorporatedi ntothepeptidogycalstructure Penicilin=competitivei nhibitoroftranspeptidase Covalenti nactivation Similarshape.Mimicsi t.Wheni tgetsi ntoactivesitei t covalentlybondstoi t&destroyse nzyme.Hasnof unction. 1moleculecanknockout1moleculeoftranspeptidase Wehavebuiltupresistancetopenicillinthereforemany bacteriali nfectionsarenol ongerstoppedbyi t ­ havemutated! Activesiterecognizesthese3( exactsamei nboth..) Penicilin Nitrogenassimilation nitrate Abundanceofe nzymei ssame. Manye nzymesneedanon ­proteinco ­factor/prostheticgroupfore nzymetowork Needsomethinge lsetobindtoe nzymeforcatalysistowork Manyco ­factors=vitamins,Heme,e tc. Nitrogenassimilation Nitratei sused Ammoniausedi nnucleicacid&aminoacidsynthesis Mutantthatdoesn'thavee nzyme.Growsonammoniaperfectlyfine.Sincedefect isBEFOREammonia(inhibitsthepathwayb4theammonia). Iftriedtogrowonnitratethenmutantdies,wildtypestillthrives/grows. Notdefectivei napoproteinsynthesisBUTi ncofactor.Can'tmake molybdenumcofactor.w/oi tcan'tmakef unctionale nzyme Nitratereductaseapoprotein. Functionale nzyme Molybdenumcofactor Energy, Enzymes & Membranes Page 6 MembraneBiology November  ­0 3  ­0 9 9:41PM Membrane=l ipids+proteins 50%+=protein Physicalbilayerbetweene nvironment&cell Hydrophilichead Lipidbilayer Hydrophobicfatty ­acidtails Amphiphathic (wholemolecule) Ex.detergents (TAIL) FLUIDITY Saturated(w/H) Linearmolecule Saturated Tailcontributestothechemistryofthemembrane Compositionaffectsmembranestructure Fluidityofmembrane Greatertheunsaturation=Higherthe fluidity(=Lowerviscosity) Influencedbytemp. Lowertemp.=Lowerthefluidity Affectstransportproteins Electrontransport RequiresEtoflowthrubilayer. Unsaturationputskinksi nthemembrane/structure Isnotholdingmax.#ofH Trans ­unsaturated Unsaturated Morespaceduetokinks Cis ­unsaturated Kinkgreaterherethan intrans V ERYDIVERSE Canhavemanydoublebonds Don'tneedjust1C=Cbond Butter=saturatedfat Solid@roomtemp. V egetable=unsaturatedfat Solidsincetheyhavehydrogenatedtheoil H2 Whysaturatefats? Increaseshelfl ife Baking Taste&texture Increasedsalevalue ­ saturatedf atsaremore expensive Chemicalprocess! Hydrogenationmaynotbecompletesincehave MANYdoublebonds Result=Partialhydrogenation Sometimesdoublebondbecomesi somerised Result=trans ­unsaturated Cisflipstotrans Increasesriskofheartdisease,e tc. BUTdatai sambiguousonwhat theydo&whythey'rebad Moresolidsincekinki nfattyacidi snotas'high' Humanshaveaconstantfluiditysincetemp.doesnot Howdoyoumaintaingrowth&e  ­ transporti ftemp.constantlychanges? Org.canadjustfluidityofmembranebyadjustingthel evelofunsaturation Enzyme:desaturase (FAMILYofe nzymes..) Saturated ­  ­ >Unsaturated FatsareALWAYSproducedsaturated( doesn'tmatterwheretheyareproduced) Enzymei ntroducesdoublebonds Org.havetheabilitytoswitche nzymeon&offtomodulatel evelsofdesaturation REMINDER: Highertemp.=higherfluidity Higherunsaturation=higherfluidity THEREFORE: @l owtemps.,needhigherunsaturationto maintainfluidity @hightemps.,needl owerunsaturationto maintainfluidity Min. AbundanceofmRNA Modulategenee xpressionquitef ast Ex.3diff.typesofDesaturase (familyofe nzymes) Energy, Enzymes & Membranes Page 7 Tails=barrierbtwni ntracellular&e xtracellularcompounds SIZE&CHARGE=MAINFACTORS Freelymovei n&out Nocharge&small O2 movesthroughbydiffusion Somecangoi n&out&somecan't H2Omovesbyosmosis Needtransportmembrane Toobig HavechargethereforeNOCHANCE... Doesn'tmatterwhati ti s Proteintransporterreq'dtotransporti t across Howcanproteinsi nteractw/hydrophobiccore? Havel otsofchargedmolecules(aminoacids) thereforehowdoesi twork? Transmembraneproteinshaveanalphahelical secondarystructure Partsthati nteractw/corehavethisstructure Alphahelixminimizeschargesofbackbone Havestretchesofnon  ­polaraminoacids Primarysequencesofproteins Puti ti ntoacomputer&i twillanalyzei t... Ifhavestretchesofnon ­polara.a.theni nteractw/ membrane ­  ­>transmembraneprotein Lookingforstretchesbetween19 ­22aminoacids Tellsyouwhetheri ti sanormal,solubleproteinori fi t interactswithamembrane Transmembrane domain Diffusion Simple Facilitated ATPbidingdomain Diffusion Entropydriven Moleculesmovedownconcentrationgradient Simple diffusion Ex.Oxygen Facilitated diffusion Pore=carrierthathelpsdiffusionoccurmorerapidly ActiveTransport ATPused(Esource)todrivetransport Againstconcentrationgradient Forcemoleculeswheretheydon'twanttogo Againste ntropy EachABCtransportertransportsdifferentthings Have100softhem Only1typeoftransporter 2differentdomains Transmembranedomain ­ alpha ­helicesthatspansthe membranethatmakesthechannelforwhichtheobjectgets transported ATPbindingdomain SHINERAMA! Asopposedtosimplediffusion. Facilitatedvs.Simplediffusion Whysohigh? Interbreedingoccurredi npop.&spreadthroughoutEurope Don'tl ivel ong.Diebeforethey're40. Notnearlyasprevalenti npeoplefromAsia& African,e tc. Energy, Enzymes & Membranes Page 8 Defect( don'thavethistransporter) SickKids ­ clonedthegene Foundthegene ­  ­>causalagent Solution:GeneTherapy Introduceawildtypecopyofgene Stillcan'tdothis... Manyalleles Deletionofphenylalanine( lossoff) Moistl ining=mucusl ayer Ciliamoveback&forth ­ clearl ungs Clearmucus&bacteriasothati tcan'tl odgethere CFTRpumpschlorideout&keepsi tmoist ABCtransporter( Chloridetransporter) ­ ATPdependent Pumpschloridei ntoe pitheliall ining Whenactivei tactivatesasodiumtransporternearby Nai stransportedi ntol ining Iontransporters Consequences:watermovesthroughmembrane(osmosis) BuildupNaClconcentrationgradientacrossmembrane THISISWHATKEEPSTHELININGMOIST... ofl ungs&i ntestinal/digestive tract water Inpatientsw/CFthisdoesn'toccur&mucusdriesout Ionbalancei sscrewedup Leadstoabuildupofbacteria Highlysusceptibletoi nfections&i nflammation Lungclearancei si nhibited Leadstodestructionofthel ung Sweatgland=oppositeorientation Sweat=saltyi nCFpatients Can'tmoveNa+ orCl  ­backi ntosweatglands FATEOF F508 ­CFTR CFTR=membraneprotein Sitsontheplasmamembrane Mustbesecretedthroughe ndoplasmicreticulumthroughthetrans  ­Golginetworktositonthe plasmamembrane Brown=wildtypeCFTR SynthesisonERribosomes Foldsi nER MovesthroughTrans  ­Golginetworktotheplasmamembrane Secretedthroughsecretarypathway ­ howtogetproteinstothemembrane F508proteincanbemadei nl ab&makei tf old( notcorrectly..bti tstillf olds) Worksokaye venw/ophenylalanine Proteincanbei nsertedi nanartificialmembrane&i tworksfine Canbedonei nmice:D BUTdoesn'tfoldcorrectly ERreq'dtomakeCFTRproteinf old ­ helpedbyCHAPERONES( helpotherproteinsf old correctly) System=ERQualitycontrol strictsystemsothatproteinsthatdon'tfoldcorrectlyaredegraded Chaperonestagproteinf ordegradationi fi tdoesn'tf oldproperly ThereforeF508 ­CFTRi sdegradedbyProteosome Workingondrugsthatdon'ttagtheproteini .e.i nhibittagging Ifthisi si nhibitedtheni tcouldmigratetomembrane&work properly Problemnotwiththeproteinbutthefactthati tdoesn'tfoldproperly Degradestaggedproteins Energy, Enzymes & Membranes Page 9 ...
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This note was uploaded on 04/12/2011 for the course BIO 1222 taught by Professor Maxwell during the Spring '08 term at UWO.

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