生物化學簡介

生物化學Biochemistry周叢照李衛(wèi)芳2010年9月8日緒論大綱：課程主要內容及要求引子：生物化學的歷史和發(fā)展趨勢教材：《生物化學》，第三版，王鏡巖等主編，2002年，高教出版社參考書：1.Biochemistry,

JeremyM.Berg,JohnL.Tymoczko,LubertStryer5theditionW.H.FreemanandCompany2.Biochemistry,DonaldVoet,JudithG.Voet.3rdedition,2003,JohnWiley&Sons,Inc.NewYork.

生物化學（上）60學時其中上課時間：54學時第一章緒論(3學時)（周叢照）第二章蛋白質(15學時)（周叢照）§2.120種氨基酸的結構和性質（2學時）§2.2蛋白質中的二級結構（1學時）§2.3蛋白質的高級結構（3學時）§2.4血紅蛋白和免疫球蛋白的結構與功能（3學時）§2.5維持蛋白質高級結構的作用力（1學時）§2.6蛋白質折疊和結構進化（2學時）§2.7蛋白質分離純化（3學時）

第三章核酸（6學時)§3.1核酸和核苷酸的結構、性質和功能（3學時）§3.2核酸的研究方法（3學時）第四章糖（3學時）§4.1糖的生物學作用（1學時）§4.2單糖和多糖（1學時）§4.3糖蛋白（1學時）第五章脂類和生物膜（6學時）§5.1脂的分類和性質（3學時）§5.2生物膜（2學時）§5.3膜蛋白（1學時）第六章酶（15學時）§6.1酶的發(fā)展歷史、作用特征及催化機理(3學時)§6.2酶的分類及酶功能的多樣性（2學時）§6.3核酶（1學時）§6.4酶促反應動力學（3學時）§6.5酶的抑制作用（3學時）§6.6別構酶及其作用原理（3學時）

第七章維生素與激素（6學時）（周叢照）§7.1維生素與輔酶（3學時）§7.2激素及其作用原理（3學時）生物化學（下）40學時其中上課時間36學時第八章代謝總論（3學時）§8.1代謝及代謝途徑的相關概念（1學時）§8.1生物能學（2學時）第九章生物膜和物質運輸（3學時）§9.1物質跨膜運輸的方式（0.5學時）§9.2小分子物質的運輸（1學時）§9.3離子載體（1學時）§9.4生物大分子的跨膜運輸（0.5學時）第十章糖酵解（6學時）§10.1糖酵解概述（1學時）§10.2糖酵解的第一階段（2學時）§10.3糖酵解的第二階段（2學時）§10.4糖酵解的調控（1學時）第十一章檸檬酸循環(huán)（3學時）§11.1檸檬酸循環(huán)的準備階段（0.5學時）§11.2檸檬酸循環(huán)的反應機制（2學時）§11.3檸檬酸循環(huán)的調控（0.5學時）第十二章生物氧化（6學時）§12.1生物氧化基本概念和呼吸鏈（1.5學時）§12.2氧化還原電位和自由能變化（0.5學時）§12.2氧化磷酸化（4學時）第十三章光合作用（3學時）§13.1光合作用概況（1學時）§13.2光合磷酸化和CO2固定（2學時）第十四章糖原的分解和生物合成（3學時）§14.1糖原的降解和糖原的生物合成（2學時）§14.2糖原代謝的調控（1學時）第十五章脂肪酸代謝（3學時）第十六章氨基酸的分解代謝（3學時）第十七章核酸代謝（3學時）WhatisBiochemistry?

thestudyofthosemoleculesusedandmanufacturedbylivingthings.Threeaspectsofbiochemistry:

1)Biochemistryisconcernedwithstructuralchemistry.Itseekstodeterminethestructuresofmoleculesfoundinlivingsystemsinordertounderstandstructure-functionrelationships.StructuralBiology2)Biochemistryisconcernedwithchemicalchange,thisisreflectedinthestudyofmetabolicpathwaysMetabolism3)BiochemistryisconcernedwithinformationwhichhasaccumulatedthroughevolutionandispreservedinDNA(orsometimesRNA).MolecularBiologyTheCentralDogmaBiochemistrythroughevolutionHowtobuildalifewithmolecules?OrTheMolecularDesignofLife

胰島素由A、B兩個肽鏈組成。人胰島素(InsulinHuman)A鏈有21個氨基酸，B鏈有30個氨基酸，共51個氨基酸組成。其中A7(Cys)-B7(Cys)、A20(Cys)-B19(Cys)四個半胱氨酸中的巰基形成兩個二硫鍵，使A、B兩鏈連接起來。此外A鏈中A6(Cys)與A11(Cys)之間也存在一個二硫鍵。人工合成胰島素1958年，中國科學院在王應睞、曹天欽、鄒承魯、鈕經義、沈昭文等先生的帶領下，提出了“世界上第一次用人工方法合成的蛋白質在中華人民共和國實現”的宏偉目標。1959年初，人工合成胰島素的工作全面展開。首先是由鈕經義、沈昭文、龔岳亭幾位研究人員組織有關人員解決了氨基酸的大量供應問題。

1965年，完成了結晶牛胰島素的合成，它有著極為深遠的意義。由于蛋白質和核酸兩類生物高分子有生命現象中所起的主要作用，人工合成了第一個具有生物活力的蛋白質，便突破了一般有機化合物領域到信息量集中的生物高分子領域之間的界限，在人類認識生命現象的漫長過程中邁出了重要的一步。合成胰島素工作的簡報發(fā)表于1965年《中國科學》。Fourtransitionsthroughevolution:1,chemicals,micromolecules2,macrobiomolecules3,energy4,stressresponseTheevolutionofliferequiredaseriesoftransitions,beginningwiththegenerationoforganicmoleculesthatcouldserveasthebuildingblocksforcomplexbiomolecules.Thenextmajortransitionintheevolutionoflifewastheformationofreplicatingmolecules.EvolutionRequires:Reproduction,Variation,SelectivePressureVariationwasintroducedbyanumberofmeans,fromsimplebasesubstitutionstotheduplicationofentiregenes.RNAappearstohavebeenanearlyreplicatingmolecule.Furthermore,someRNAmoleculespossesscatalyticactivity.However,therangeofreactionsthatRNAiscapableofcatalyzingislimited.Withtime,thecatalyticactivitywastransferredtoproteins,linearpolymersofthechemicallyversatileaminoacids.RNAdirectedthesynthesisoftheseproteinsandstilldoesinmodernorganismsthroughthedevelopmentofageneticcode,whichrelatesbasesequencetoaminoacidsequence.Eventually,RNAlostitsroleasthegenetothechemicallysimilarbutmorestablenucleicacidDNA.Inmodernorganisms,RNAstillservesasthelinkbetweenDNAandprotein.EnergyTransformationsAreNecessarytoSustainLivingSystemsATPservesasthecellularenergycurrencythatlinksenergy-yieldingreactionswithenergy-requiringreactions.ATPitselfisaproductoftheoxidationoffuelmolecules,suchasaminoacidsandsugars.Withtheevolutionofmembraneshydrophobicbarriersthatdelineatethebordersofcellsiongradientswererequiredtopreventosmoticcrises.ThesegradientswereformedattheexpenseofATPhydrolysis.Later,iongradientsgeneratedbylightortheoxidationoffuelmoleculeswereusedtosynthesizeATP.

CellsCanRespondtoChangesinTheirEnvironments

Thefinaltransitionwastheevolutionofsensingandsignalingmechanismsthatenabledacelltorespondtochangesinitsenvironment.Thesesignalingmechanismseventuallyledtocell-cellcommunication,whichallowedthedevelopmentofmore-complexorganisms.Therecordofmuchofwhathasoccurredsincetheformationofprimitiveorganismsiswritteninthegenomesofextantorganisms.(molecularevolution)HistoryofBiochemistry(upto1982)

1835JonsBerzelius

chemicalcatalysis,usesamylase(淀粉酶)asanexample.1859CharlesDarwinpublishes

OntheOriginofSpecies.LouisPasteur

fermationcatalyzedbyenzymes,"essence"ofyeast.1865GregorMendelpublisheshis

theoryofgenetics.1869FredrickMeischerdiscovers

DNAincellnuclei.EduardandHansBuchnerextractsmaterielfromyeast,conversionofglucosetoalcohol.

1914FritzLipmann,theroleofATPinenergymetabolism.1926JamesSumner,

crystallinejackbean(刀豆)urease,isaprotein.1926ThomasHuntMorganwrites

TheTheoryoftheGene.1934ArnoldBeckmandevelopesthefirstpHmeter.1937HansKrebsdiscoversthecitricacidcycle(TCAcycle).1941GeorgeBeadle&EdwardTatum,theone-gene,one-enzymehypothesis.OswaldAvery,ColinMacLeod,andMaclynMcCarthy

DNAisthegeneticmaterial.

1950EdwinChargaff

A=T,G=C(Chargaff'srules).1952LinusPaulingandRobertCorey

α-helixandtheβ-pleatedsheet1953JamesWatsonandFrancesCrickthedoublehelixmodelofDNA.1953FredrickSangerthefirstaminoacidsequenceofaprotein(insulin).1956EarlSutherlandisolatescyclicAMP.1957MatthewMeselsonandFranklinStahl

semiconservativeDNAreplication.

1960JohnKendrewandMaxPertuzobtain

thefirst3-Dstructureofproteins

(hemoglobinandmyoglobin).1960JeraldHuritzandSamuelWeissdiscover

RNApolymerase.1961FrancoisJacobandJaquesMonodpropound

theoperonmodelofgenecontrol.1963Jean-PierrreChanguex,F.Jacob,andJ.Monod

Allostericmodelforinhibitionofenzymes1964Severalgroups

Acrylamidegelelectrophoresisofproteinsisdeveloped

MarshalNirenberg,H.GobindKhorana,andSeveroOchoacompletetheelucidationofthegeneticcode1965DavidPhillips

3-Dmodeloffirstenzyme(lysozyme)1965RobertHolleydetermines

thestructureofatransfer-aaRNA.1965JeromeVinograddiscovers

superhelicaltwisting.1968MarkPtashneandWalterGilbertidentify

thefirstrepressorgenes1969Firstsynthesisofanenzyme(ribonuclease).

1970HamiltonSmithdiscovers

restrictionendonucleases.1970HowardTeminandDavidBaltimorediscover

reversetranscriptase.1973StanleyCohenandHerbertBoyerprepare

recombinantDNA.1974Sung-HouKim,etal.produce

thefirstX-raystructureoftransferRNA.1977CesarMilsteindiscovershowtoproduce

monoclonalantibodies.1977AllanMaxamandWalterGilbertdevelop

achemistryforsequencingDNA.1977FredrickSanger,S.NicklenandA.R.Coulsondevelop

achemistryforsequencingDNA.1977PhillipSharpandRichardRobertsdiscover

introns(interveningsequences).1982AmzelLM,McKinneyM,NarayananP,PedersenPL

Firstx-raystructureofamembraneprotein(9?).1980sandearly1990sWonderfultimeforMolecularBiologyHumanGenomeProject(1988-)CentralDogma(CrickF.1958)MolecularBiologyOmics:

MetabolomicsTranscriptomicsProteomics

StructuralGenomics

(1998-)

GenomicsTheeraofOmics:(1998-2003orlater?)Omics=Oh!Mix!(millenniumchaos?)Whatis

StructuralGenomics?Itisanapproachaimingatsolving

3-Dstructures

oftheproteinsencodedbyan

entiregenome.

KimSH.NatureStructuralBiology1998OriginalgoalsofSG:1,toestablishacatalogue/libraryofallfoldscoveringtheentireproteinuniverse2,tohelptheannotationofsequencedgenomesInternationalStructuralGenomicsProjectsUSAandCanada:(1998-)9NIHStructuralGenomicsCentersEU:France(3centers:Paris-Sud,Marseille,Strasbourg)Germany(StructuralGenomicsFactory)UK(2centers)(SPINE:StructuralProteomicsinEurope,2001-)Asia:Japan(RIKEN…)China(Yun-YuSHI;ZiheRAO…)(2002-)Korea:Israel:WeizmannStructuralProteomicsCenterFlowchart:1,Targetsselection:Bioinformatics2,PCRandcloning:MolecularBiology3,Proteinproduction:Biochemistry4,Datacollection(X-raydiffraction/NMR)5,Structuredetermination:StructuralBiology6,Functioninterpretation:GeneralBiologyCorecharacteristics:Highthroughput(hundredstothousandssamples)Large-scale(milligramlevelofproteinsample)Multi-disciplineintegration(frommolecularbiologytostructuralbiology)highthroughput

parallelization(平行化)

miniaturization(微量化)

automation(自動化)cloningrobot:500-1000clones/3dayscrystallizationrobot:96x3drops/5minproteinsampleforcrystallizationtrialsinnanolitervolumes(<10nl)HansenCL.etal.PNAS;2002TagSize(aa)ApplicationSequenceorGenBankaccessionno.His6--10affinityHHHHHH(HH)T711affinityASMTGGQQMGRS15affinityKETAAAKFERQHMDSArg1,5,6affinityRRRRR(R)CBP26affinityKRRWKKNFIAVSAANRFKKISSSGALFLAG4or8affinityDYKDorDYKDDDDKStrep8affinityWSHPQFEKNusA491SET,affinityAccessionno.AAN82367MBP396SET,affinityAccessionno.AAC43128GST220SET,affinityAccessionno.AAB59203ZZ116SET,affinityAccessionno.M74186TrxA109SETAccessionno.AAC40210Gb156SETAccessionno.1MPEADsbADsbC208236SETSETAccessionno.P24991Accessionno.P21892Differenttagswillmakethedifference

DifferentE.colistrains(DE3series)

BL21(DE3)BL21-pLysSBL21-codon-plusTunerRosettaRosetta-pLysSGoldStarCo-expressionwithchaperones(DnaK-DnaJ-GrpEand/orGroEL-GroES)

Mainlyworksforthosepartiallysolubletargets

Co-purificationofchaperoneswiththetargetprotein

Re-aggregation/precipitationafterremovalofchaperonesEukaryoticexpressionsystems(post-translationalmodifications)PichiapastorisSaccharomycescerevisiaeInsect

cells(Sf9、SF21)Mammaliancells(CHOetal.)Virus-mediatedQuantityandCost-effectivityDNAshufflingSaturationmutagenesisError-pronePCR…Toimprove:

thesolubility,activityand/orstabilityReetz,PNAS;2004invitro/directedevolutionProteinssamplesforcrystallizationtrials:1,pureandhomogenous2,stableinsolutionoflowsaltconcentration,properpHandreductant(BME,DTTorTCEP)3,athighconcentration(normally~10mg/ml)HangingdropSittingdropProteinsamplesforNMRdatacollection:1,doublelabelingwith13Cand15N2,nooligomerizationoraggregation3,stableforatleastoneweekatRTor4oC4,highexpressionlevel(cost-effective)Optimizationofxtals:1,abouthalfofthextalswillnotdiffractat3?orhigherresolution2,parameters:saltconcentration,buffersystem,pH,divalentortrivalentirons3,precipitants:PEGs,MPD,salt,alcohol,…4,ligandsorsubstrates/products/analogs5,proteinornucleicacidpartnersDatacollection:X-raydiffraction:in-houseX-raygeneratorsynchrotronradiationacceleratorNMRspectrometry:500mHz,600mHz,800mHz900mHzin-houseX-raygeneratorDatacollection:synchrotronradiationaccelerator

Assignmentoffunctionbasedonstructuralsimilarity

Reciprocalstimulationandvalidation:biochemicalassaysversusstructureanalyses

Structure-directeddrugdesign

Functioninterpretationandapplication:

Assignmentoffunctionbasedonstructuralsimilarity3-DstructureDALIcomparisonCloseststructuresSimilarmolecularfunction?http://www.ebi.ac.uk/daliHolmL,KaariainenS,RosenstromP,SchenkelA.(2008)SearchingproteinstructuredatabaseswithDaliLitev.3.

Bioinformatics24,2780-2781.

Reciprocalstimulationandvalidation:biochemicalassaysvs.structurea