2011浙大考研本科生物化學(xué)課件SUMMARY1

Whatisbiochemistry?Biochemistryorbiologicalchemistryisthebranchofsciencedealingwiththechemicalcompounds,reactions,andotherprocessesthatoccurinlivingorganisms.Whatisthebasicgoalofthescienceofbiochemistry?Thebasicgoalofthescienceofbiochemistryistodeterminehowthecollectionsofinanimatemoleculesthatconstitutelivingorganismsinteractwitheachothertomaintainandperpetuatelife.Biochemistryseekstodescribeinmoleculartermsthosestructures,mechanisms,andchemicalprocessessharedbyallorganismsandtodiscovertheorganizingprinciplesthatunderlielifeinallofitsdiverseforms.Whatdistinguisheslivingorganismsfrominanimateobjects?Livingorganismsarestructurallycomplicatedandhighlyorganized.Theypossessintricateinternalstructures.LivingOrganismsextract,transform,anduseenergyfromtheirenvironment,usuallyintheformofeitherchemicalnutrientsortheradiantenergyofsunlight.Themostcharacteristicattributeoflivingorganismsisthecapacityforpreciseself-replicationandself-assembly,apropertythatcanberegardedasthequintessenceofthelivingstate.Catabolism:Thepathwaydegradeorganicnutrientsintosimpleendproductsinordertoextractchemicalenergyandconvertitintoaformusefultothecell.Anabolism:Thepathwaystartwithsmallprecursormoleculesandconvertthemtolargerandmorecomplexmoleculesandrequiretheinputofenergy.Homogenizationinabout0.2MsucroseOrganellesCanBeIsolatedbyCentrifugationConfiguration:Thearrangementoftheatomsofamoleculeinspace,withoutregardtoarrangementsthatdifferonlyasafterrotationaboutoneormoresinglebonds.Conformation:Thevariousarrangementsoftheatomsofamoleculeinspacethatdifferonlyasafterrotationaboutsinglebonds.FiveGeneralTypesofChemicalTransformationsOccurinCellsa;functional-grouptransfersb;Oxidations-reductionsc;Internalrearrangementsd;Cleavageandformationofcarbon-carbonbondse;CondensationreactionCellsHaveaStructuralHierarchyThereisastructuralhierarchyinthemolecularorganizationofcells.Cellscontainorganelles,suchasnuclei,mitochondria,andchloroplasts,whichinturncontainsupramolecularcomplexes,suchasmembranesandribosomes,andtheseconsistinturnofclustersofmacromoleculesthatareboundtogetherbymanyrelativelyweak,noncovalentforces.Themacromoleculesconsistofcovalentlylinkedsubunits.RNAorRelatedPrecursorsMayHaveBeentheFirstGenesandCatalystsOnepossible"RNAworld"scenario,showingthetransitionfromtheprebioticRNAworld(shadesofyellow)tothebioticDNAworld(orange).

RNAMoleculesMayHaveBeentheFirstGenesandCatalystsFourtypesofweakinteractionsoccurwithinandbetweenbiomoleculesinanaqueoussolvent:hydrogenbondsandionic,hydrophobic,andvander

Waalsinteractions.Althoughweakindividually,theseinteractionscollectivelycreateaverystrongstabilizingforceforproteins,nucleicacids,andmembranes.Weak(noncovalent)interactionsarealsoattheheartofenzymecatalysis,antibodyfunction,andreceptor-ligandinteractions.Nonpolar,AliphaticRGroupsThehydrocarbonRgroupsarenonpolarandhydrophobic.Thesidechainsofalanine,valine,leucine,andisoleucine,withtheirdistinctiveshapes,areimportantinpromotinghydrophobicinteractionsandtendtoclustertogetherwithinproteinstructures.Glycinehasthesimplestaminoacidstructure.Whereitispresentinaprotein,theminimalsterichindranceoftheglycinesidechainallowsmuchmorestructuralflexibilitythantheotheraminoacids.Proline

hasadistinctivecyclicstructure.Thesecondaryamino(imino)groupofProresiduesisheldinarigidconformationthatreducesthestructuralflexibilityoftheproteinatthatpoint.AminoAcidsCanBeClassifiedbyRGroupAromaticRGroupsPhenylalanine,tyrosine,andtryptophan,withtheiraromaticsidechains,arerelativelynonpolar(hydrophobic).Allcanparticipateinhydrophobicinteractions,whichareparticularlystrongwhenthearomaticgroupsarestackedononeanother.Thehydroxylgroupoftyrosinecanformhydrogenbonds,anditactsasanimportantfunctionalgroupintheactivityofsomeenzymes.Tyrosineandtryptophanaresignificantlymorepolarthanphenylalaninebecauseofthetyrosinehydroxylgroupandthenitrogenofthetryptophan

indolering.Tryptophanandtyrosine,andtoalesserextentphenylalanine,absorbultravioletlight.Thisaccountsforthecharacteristicstrongabsorbanceoflightbyproteinsatawavelengthof280nm,andisapropertyexploitedbyresearchersinthecharacterizationofproteins.

Asparagineandglutaminearetheamidesoftwootheraminoacidsalsofoundinproteins,aspartateandglutamate,respectively,towhichasparagineandglutamineareeasilyhydrolyzedbyacidorbase.CysteinehasanRgroup(athiolgroup)thatisapproximatelyasacidicasthehydroxylgroupoftyrosine.Cysteinerequiresspecialmentionforanotherreason.Itisreadilyoxidizedtoformacovalentlylinkeddimericaminoacidcalledcystine,inwhichtwocysteinemoleculesarejoinedbyadisulfidebridge.Disulfidebridgesofthiskindoccurinmanyproteins,stabilizingtheirstructures.Polar,UnchargedRGroupsTheRgroupsoftheseaminoacidsaremoresolubleinwater,orhydrophilic,thanthoseofthenonpolaraminoacids,becausetheycontainfunctionalgroupsthatformhydrogenbondswithwater.Thisclassofaminoacidsincludesserine,threonine,cysteine,methionine,asparagine,andglutamine.Thepolarityofserineandthreonineiscontributedbytheirhydroxylgroups;thatofcysteineandmethioninebytheirsulfuratom;andthatofasparagineandglutaminebytheiramidegroups.CysteinehasanRgroup(athiolgroup)thatisapproximatelyasacidicasthehydroxylgroupoftyrosine.Cysteinerequiresspecialmentionforanotherreason.Itisreadilyoxidizedtoformacovalentlylinkeddimericaminoacidcalledcystine,inwhichtwocysteinemoleculesarejoinedbyadisulfidebridge.Disulfidebridgesofthiskindoccurinmanyproteins,stabilizingtheirstructures.PositiuelyCharged(Basic)RGroupsTheaminoacidsinwhichtheRgroupshaveanetpositivechargeatpH7.0arelysine,whichhasasecondaminogroupatthepositiononitsaliphaticchain;arginine,whichhasapositivelychargedguanidinogroup;andhistidine,containinganimidazolegroup.HistidineistheonlystandardaminoacidhavingasidechainwithapKanearneutrality.NegatiuelyCharged(Acidic)RGroupsThetwoaminoacidshavingRgroupswithanetnegativechargeatpH7.0areaspartateandglutamate,eachwithasecondcarboxylgroup.Theseaminoacidsaretheparentcompoundsofasparagineandglutamine,respectively.AminoAcidsHaveCharacteristicTitrationCurvesThetitrationcurveof0.1Mglycineat25C.Theionicspeciespredominatingatkeypointsinthetitrationareshownabovethegraph.Theshadedboxes,centeredaboutpK1=2.34andpK2=9.60,indicatetheregionsofgreatestbufferingpowerThereAreSeveralLevelsofProteinStructureAdescriptionofallcovalentbonds(mainlypeptidebondsanddisulfidebonds)linkingaminoacidresiduesinapolypeptidechainisitsprimarystructure.Themostimportantelementofprimarystructureisthesequenceofaminoacidresidues.Secondarystructurereferstoparticularlystablearrangementsofaminoacidresiduesgivingrisetorecurringstructuralpatterns.Tertiarystructuredescribesallaspectsofthethree-dimensionalfoldingofapolypeptide.Whenaproteinhastwoormorepolypeptidesubunits,theirarrangementinspaceisreferredtoasquaternarystructure.Ion-exchangechromatography;exploitsdifferencesinthesignandmagnitudeofthenetelectricchargesofaproteinsatagivepH.Thecolumnmatrixasyntheticpolymercontainingboundchargegroupsarecalledcationexchanger.TheseparationcanbeoptimizedbygraduallychangingthepHand/orsaltconcentration.Size-exclusionchromatography;alsocalledgelfiltration.Thismethodseparatesproteinsaccordingtosize.Thecolumncontainsacross-linkedpolymerwithporesofselectedsize.Largerproteinsmigratefasterthansmallerones,becausetheyaretoolargetoentertheporesinthebeadsandhencetakeamoredirectroutethroughthecolumn.Thesmallerproteinsentertheporesandareslowedbythemorelabyrinthianpaththeytakethroughthecolumn.Affinitychromatographyseparatesproteinsbytheirbindingspecificities.Theproteinsretainedonthecolumnarethosethatbindspecificallytoaligandcross-linkedtothebeads.(Inbiochemistry,theterm"ligand"isusedtorefertoagroupormoleculethatisbound.)Afternonspecificproteinsarewashedthroughthecolumn,theboundproteinofparticularinterestiselutedbyasolutioncontainingfreeligand.Ion-exchangechromatographySize-exclusionchromatographyAffinitychromatographyTwo-DimensionalElectrophoresisTheAminoAcidSequencesofNumerousProteinsHaveBeenDeterminedTherelationshipbetweenaminoacidsequenceandbiologicalfunction.Proteinswithdifferentfunctionsalwayshavedifferentaminoacidsequences.Iftheprimarystructureisaltered,theproteinfunctionmayalsochanged.Oncomparingfunctionallysimilarproteinsfromdifferentspecies,thoseproteinsoftenhavebeenfoundtohavesimilaraminoacidsequences.ShortPolypeptidesAreSequencedUsingAutomatedProceduresFrederrickSangerPehr

EdmanDeterminationofaminoacidsequenceofpeptidesandproteinsIsolationandpurificationofpeptidesAnalysisofthepeptidemass(massspectrometryandSDSelectrophoresis)BreakingDisulfidebondsPurificationofthepeptideswithcleaveddisulfidebondsN-terminalaminoacidsequenceanalysisofthepeptidesCleavingthepeptidesPurifyingthedigestsSequencingofeachpurifiedpeptidefragmentsOrderingthepeptidefragmentsLocationdisulfidebondsFiveconstraintsaffectthestabilityofanαhelix:theelectrostaticrepulsion(orattraction)betweenaminoacidresidueswithchargedRgroups(2)thebulkinessofadjacentRgroups(3)theinteractionsbetweenaminoacidsidechainsspacedthree(orfour)residuesapart(4)theoccurrenceofProresidues(5)theinteractionbetweenaminoacidsattheendsofthehelixandtheelectricdipoleinherenttothisstructure.β-conformation;Anextended,zigzagarrangementofapolypeptidechain.

?sheet;Thezigzagpeptidechainscanbearrangedsidebysidetoformastructureresemblingaseriesofplates.

?turns;Atypeofsecondarystructureinpolypeptidesconsistingoffouraminoacidresiduesarrangedinatightturn(180o)sothatthepolypeptideturnbackonitself.Motifs(SupersecondaryStructures);

Inproteins,aunitexhibitingaparticularthree-dimensionalstructurethatisfoundinavarietyofproteinsandusuallyisassociatedwithaparticularfunction(helix-loop-helix,zincfinger)Structuraldomains;Polypeptidewithmorethanafewhundredaminoacidresiduesoftenfoldintotwoormorestable,globularunitscalledstructuraldomins.Denaturation;Alossofthree-dimensionalstructuresufficienttocauselossoffunctioniscalledproteindenaturation(byheat,extremesofpH,organicsolvents,urea,guanidinehydrochloride,ordetergents).Certainglobularproteinsdenaturedbyheat,extremesofpH,ordenaturingreagentswillregaintheirnativestructureandtheirbiologicalactivity,iftheyarereturnedtoconditionsinwhichthenativeconformationisstable,thisprocesscalledrenaturation.Thermodynamically,thefoldingprocesscanbeviewedasakindoffree-energyfunnel.Theunfoldedstatesarecharacterizedbyahighdegreeofconformationalentropyandrelativelyhighfreeenergy.Asfoldingproceeds,thenarrowingofthefunnelrepresentsadecreaseinthenumberofconformationalspeciespresent.Smalldepressionsalongthesidesofthefree-energyfunnelrepresentsemistableintermediatesthatcanbrieflyslowthefoldingprocess.Atthebottomofthefunnel,anensembleoffoldingintermediateshasbeenreducedtoasinglenativeconformation.Molecularchaperones;areproteinsthatinteractwithpartiallyfoldedorimproperlyfoldedpolypeptides,facilitatingcorrectfoldingpathwaysorprovidingmicroenvironmentsinwhichfoldingcanoccur(heatshockprotein).Chaperonins;areelaborateproteincomplexesrequiredforthefoldingofanumberofcellularproteinsthatdonotfoldspontaneously.Ligand;Amoleculeboundreversiblybyaproteiniscalledaligand.Bindingsite;Aligandbindsatasiteontheprotein,whichiscomplementarytotheligandinsize,shape,chargeandhydrophobicorhydrophiliccharacter.Inducedfit;Thebindingofaproteinandligandisoftencoupledtoaconformationalchangeintheproteinthatmakesthebindingsitemorecomplementarytotheligand,permittingtighterbinding.Thisstructuraladaptationiscalledinducedfit.Substrate;Themoleculeacteduponbyenzymeiscalledtobereactionsubstrate.Antibodies(immunoglobulins,Ig);Theproteinsattheheartofthehumoralimmuneresponsearesolubleandbindbacteria,viruses,orlargemoleculesidentifiedasforeignandtargetthemfordestruction.IgareproducedbyBlymphocytesandmakingup20%ofbloodprotein.ImmunoglobulinG(IgG)isthemarjorclassofantibodymoleculeandoneofthemostabundantproteinsinthebloodserum.IgGhasfourpolypeptidechains:twolargeones,calledheavychains,andtwolightchains,linkedbynoncovalentanddisulfidebondsintoacomplexofMr150,000.Antigen;Anymoleculeorpathogencapableofelicitinganimmuneresponseiscalledantigen.Anantigenmaybeavirus,abacteriacellwall,oranindividualproteinorothermacromolecule(Mr>5,000).Polyclonalantibodies;arethoseproducedbymanydifferentBcllesrespondingtooneantigen,suchasaproteininjectedintoananimal.CellinthepopulationofBcellsproduceantibodiesthatbindspecific,differentepitopeswithintheantigen.Thus,polyclonalpreparationscontainamixtureofantibodiesthatrecognizedifferentpartsoftheprotein.Monoclonalantibodies;aresynthesizedbyapopulationofidenticalBcells(aclone)grownincellculture.Theseantibodiesarehomogeneous,allrecognizingthesameepitope.ELISA(enzyme-linkedimmunosorbentassay)1.Coatsurfacewithsample(antigens)2.Blockingwithnonspecificprotein3.Incubatewithantibody4.Incubatewithsecondaryantibody(antibody-enzymecomplex)5Addsubstrate6.FormationofcoloredImmunoblotassay(WesternBlotting)1.Separationofproteinsbygelelectrophoresis2.Transfertheseparatedproteinstoamembrane3.Membraneblockingwithnonspecificprotein4.Incubatewithsecondaryantibody(antibody-enzymecomplex)5.Addsubstrate6.FormationofcoloredMyosinThickFilamentsSlidealongActinThinFilamentsATPbindstomyosin,andacleftinthemyosinmoleculeopens,disruptingtheactin-myosininteractionsothattheboundactinisreleased.ATPisthenhydrolyzed,causingaconformationalchangeintheproteintoa“high-energy”statethatmovesthemyosinheadandchangesitsorientationinrelationtotheactinthinfilament.AsthephosphateproductofATPhydrolysisisreleasedfrommyosin,anotherconformationalchangeoccursinwhichthemyosincleftcloses,strengtheningthemyosin-actinbinding.4.A“powerstroke”duringwhichtheconformationofthemyosinheadreturnstotheoriginalrestingstate,itsorientationrelativetotheboundactin.ADPisthenreleasedtocompletethecycle.EnzymesAreClassifiedbytheReactionsTheyCatalyzeSpecificityofEnzymes:Chemicalreactionsofmanytypestakeplacebetweensubstratesandenzymefunctionalgroups(specificaminoacidsidechains,metalions,andcoenzymes).Catalyticfunctionalgroupsonanenzymemayformatransientcovalentbondwithasubstrateandactivateitforreaction,orsomegroupmaybetransientlytransferredformthesubstratetoagroupontheenzyme.CatalyticPowerofEnzyme:Bindingenergyisamajorsourceoffreeenergyusedbyenzymestolowertheactivationenergiesofreactions.1.Muchofthecatalyticpowerofenzymesisultimatelyderivedfromthefreeenergyreleasedinformingmultipleweakbondsandinteractionsbetweenanenzymeanditssubstrate.Thisbindingenergycontributestospecificityaswellascatalysis.2.Weakinteractionsareoptimizedinthereactiontransitionstate;enzymeactivesitesarecomplementarynottothesubstratesperse,buttothetransitionstatesthroughwhichsubstratespassastheyareconvertedintoproductsduringthecourseofanenzymaticreaction.TheRelationshipbetweenSubstrateConcentrationandReactionRateCanBeExpressedQuantitativelyMichaelisConstant,KmandMichaelis-MentenEquation:E+SESE+PV0=k2[ES]K1K-1K2RateofESformation=k1([Et]-[ES])[S]RateofESbreakdown=k-1[ES]+k2[ES]K1([Et]–[ES])[S]=k-1[ES]+k2[ES]K1[Et][S]–k1[ES][S]=(k1+k2)[ES]K1[Et][S]=(k1[S]+k-1+k2)[ES][ES]=[ES]=k1[S]+k-1+k2K1[Et][S][Et][S][S]+(k2+k-1)/k1(k2+k-1)k1=Km;MichaelisConstant[ES]=V0=V0=WhenV0=1/2Vmax,Km=[S],KmisequivalenttothesubstrateconcentrationatwhichV0isone-halfVmax[Et][S]Km+[S]k2[Et][S]Km+[S]Vmax[S]Km+[S]TheRegulatoryStepinManyPathwaysIsCatalyzedbyanAllostericEnzymeFeedbackInhibition;Theregulatoryenzymeisspecificityinhibitedbytheendproductofthepathwaywhenevertheconcentrationoftheendproductexceedsthecell’srequirements.PhosphorylGroupAffecttheStructureandCatalyticActivityofProteinsProteinkinases:Theattachmentofphosphorylgroupstospecificaminoacidresiduesofaproteiniscatalyzedbyproteinkinases.DNAMoleculesHaveDistinctiveBaseCompositionChargaffsrules;ThebasecompositionofDNAgenerallyvariesfromonespeciestoanotherDNAspecimensisolatedfromdifferenttissuesofthesamespecieshavethesamebasecomposition.ThebasecompositionofDNAingivenspeciesdoesnotchangewithanorganism’sage,nutritionalstate,orchangingenvironment.InallcellularDNAs,regardlessofthespecies,thenumberofadenosineresiduesisequaltothenumberofthymidineresidues(A=T),andthenumberofguanosineresiduesisequaltonumberofcytidineresidues(G=C).Fromtheserelationshipsitfollowsthatthesumofthepurineresiduesequalsthesamofthepyrimidineresidues(A+G=T+C).NucleicAcidStructureFromx-raydiffractionstudiesofDNAfibersandthebaseequivalencesinDNAdiscoveredbyChargaff(A=TandG≡C),WatsonandCrickpostulatedthatnativeDNAconsistsoftwoantiparallelchainsinaright-handeddouble-helicalarrangement.Complementarybasepairs,A=TandG≡C,areformedbyhydrogenbondingwithinthehelix,andthehydrophilicsugar-phosphatebackbonesarelocatedontheoutside.Thebasepairsarestackedperpendiculartothelongaxis,0.34nmapart;thereareabout10basepairsineachcompleteturnofthedoublehelix.LongDNASequencesCanBeDeterminedLongDNASequencesCanBeDeterminedThebiologicalfunctionsofthelipids:Energystorage(fatsandoils)Majorstructuralelementsofbiologicalmembrane(phospholipidsandsterols).Others(enzymecofactors,light-absorbingpigments,hormone,electroncarrier)Threegeneraltypesofmembranelipids:Glycerophospholipids,inwhichthehydrophobicregionsarecomposedoftwofattyacidsjoinedtoglycerol;Sphingolipids,inwhichasinglefattyacidisjoinedtoafattyamine,sphingosine;Sterols,compoundscharacterizedbyarigidsystemoffourfusedhydrocarbonrings.Theprostaglandins(PG)containafive-memberedringofcarbonatomsoriginallypartofthechainofarachidonicacid.Theyderivetheirnamefromthetissueinwhichtheywerefirstrecognized(theprostategland).Theprostaglandinsaffectawiderangeofcellularandtissuefunctions.Someprostaglandinsstimulatecontractionofthesmoothmuscleoftheuterusduringlaborormenstruation.Othersaffectbloodflowtospecificorgans,thewake-sleepcycle,andtheresponsivenessofcertaintissuestohormonessuchasepinephrineandglucagon.Prostaglandinsinathirdgroupelevatebodytemperature(producingfever)andcauseinflammation,resultinginpain.Vitamins:Thecompoundsthatareessentialtothehealthofhumansandothervertebratesbutcannotbesynthesizedbytheseanimalsandmustbethereforebeobtainedinthediet.VitaminsAandDAreHormonePrecursorsVitaminDisaderivativeofcholesterolandtheprecursortoahormoneessentialincalciumandphosphatemetabolisminvertebrateanimals.VitaminD3,alsocalledcholecalciferol,isnormallyformedintheskininaphotochemicalreactiondrivenbytheultravioletcomponentofsunlight.Itisalsoabundantinfishliveroils,andisaddedtocommercialmilkasanutritionalsupplement.VitaminD3itselfisnotbiologicallyactive,butitistheprecursorof1,25-dihydroxycholecalciferol,apotenthormonethatregulatestheuptakeofcalciumintheintestineandthebalanceofreleaseanddepositionofbonecalciumandphosphate.VitaminA(retinol)isapigmentessentialtovision.DeficiencyofvitaminAleadstoavarietyofsymptomsinhumansandexperimentalanimals,whichincludedryskin,dryeyes,drymucousmembranes,retardeddevelopmentandgrowth,sterilityinmale,andnightblindness,anearlysymptomcommonlyusedinthemedicaldiagnosisofvitaminAdeficiency.VitaminsAandDAreHormonePrecursorsVitaminEisthecollectivenameforagroupofcloselyrelatedlipidscalledtocopherols,allofwhichcontainasubstitutedaromaticringandalonghydrocarbonsidechain.Tocopherolsarefoundinhens'eggsandvegetableoils,andareespeciallyabundantinwheatgerm.DeficiencyofvitaminEisveryrareinhumans,butwhenlaboratoryanimalsarefeddietsdepletedofvitaminE,theydevelopscalyskin,muscularweaknessandwasting,andsterility.VitaminKisalipidcofactorrequiredfornormalbloodclotting.VitaminKlisfoundingreenplantleaves,andarelatedform,vitaminK2(menaquinone),isformedbybacteriaresidingintheanimalintestine.Thevitaminactsintheformationofprothrombin,abloodplasmaprot