1、BiochemistryFor Speciality of Biotechnology or Bioengineering at CSUMr.XIA JinlanChapter 14 Enzyme kinetics n Introduction to enzymes 酶（学）导论n Factors affecting enzyme activity 影响酶活性的因素n Enzyme assaysn Enzyme kinetics and inhibition 酶动力学和抑制作用Introduction to enzymes 酶（学）导论酶（学）导论nThe features of enzyme
2、s 酶的特征nSome terms of enzyme 酶的几种术语nClasses of enzymes 酶的种类nEnzyme Nomenclature 酶的命名法nTraditional nomenclature 传统命名法nSystematic nomenclature 系统命名法nExpression of enzyme activity 酶活性的表示nComponents of enzyme 酶的成分nCofactors 辅助因子nPowerful Catalytic power 强的催化力强的催化力nIncrease reaction rate up to 1016 foldnM
3、ake reaction taken place under relatively mild conditions of temperature and pHnHigh specificity 高的特异性高的特异性nTo the substrates that they act on and the products they formnEnzyme activity can be regulated 酶活性可以酶活性可以被调控被调控nBasically are proteins,except few ribozymes 除除了少量的核酶外了少量的核酶外,基本上是蛋白质基本上是蛋白质 Enzy
4、mes have four basic features酶的四个基本特征酶的四个基本特征Some terms of enzymes 酶的几种术语酶的几种术语nProteoenzyme蛋白酶nRibozyme核酶nAbzyme 抗体酶nHoloenzyme全酶nApoenzyme脱辅酶nMultimeric enzyme多聚合体酶(多亚基酶)Enzymes are classified into six classes based on the type of reaction catalyzed基于催化反应的类型基于催化反应的类型,酶可分为六种类型酶可分为六种类型nOxidoreductase
5、s 氧化还原酶nTransfer of electrons 电子的转移nTransferases 转化(或转移)酶nTransfer of functional groups 分子间功能基团的转移nHydrolases 水解酶nHydrolysis reactions 水解反应nLyases 裂解酶nCleavage of C-C,C-O,C-N etc.,often forming a double bond C-C,C-O,C-N键的裂解,并通常形成双键的反应nIsomerases 异构酶nTransfer of groups within a molecule 分子内基团的转移nLiga
6、ses(or synthases)链接酶(或合成酶)nBond formation coupled to ATP hydrolysis伴随ATP水解的键合Enzymes are classified into six classes based on the type of reaction catalyzed NameType of reaction catalyzed ExampleOxido-reductasesTransfer of electronsA-+B A+B-Alcohol dehydrogenaseTransfer-asesTransfer of functional gr
7、oupsA-B+CA+B-CHexokinaseHydrolasesHydrolysis reactionsA-B+H2OA-H+B-OHTrypsinLyasesCleavage of C-C,C-O,C-N etc.,often forming a double bondPyruvate decarboxylaseIsomerasesTransfer of groups within a moleculeMaleate isomaseLigases(or synthases)Bond formation coupled to ATP hydrolysisPyruvate carboxyla
8、seA+B A-BX-A-B-Y Y-A-B-XX-A-B-Y A=B+Y-XTraditional Nomenclature of enzymeshexokinase己糖激酶urease尿酶fructose 1,6-bisphosphatase果糖 1,6-二磷酸酶1.Named after substrate by adding aseName ofsubstrate+type ofenzyme+aseName ofsubstrate+asepyruvate decarboxylase丙酮酸脱羧酶Maleate isomerase马来酸异构酶3.Named after source and
9、 propertiesTrypsin 胰蛋白酶Chymotrypsin 胰凝乳蛋白酶2.Named after amino acid residue in the active site by adding aseSerine proteases 丝氨酸蛋白(水解)酶Systematic nomenclature:Each enzyme is uniquely identified by a four-digit classification number nEnzyme commission(EC)number:nFour-digit classification numbernWhat d
10、o these numbers signify?Trypsin 3.4.21.4hydrolaseproteaseserine protease4th enzyme of this classTrypsin EC 3.4.21.4Chymotrypsin EC 3.4.21.1Elastase EC 3.4.21.36Expression of enzyme activity 酶活性的表示酶活性的表示 nInitial rate(V0)of the reactionn mmol of substrate transformed per minute 每分钟转化底物的微摩尔数nV0:X mmol
11、.min-1 nEnzyme unit 酶的单位酶的单位,SI unitnThe amount that catalyzes the formation of one mmol of product per minute 每分钟催化1mmol产物的形成所需的酶量nU:mmol.min-1nKatal,SI unitnAmount of enzyme catalyzing the conversion of one mole of substrate to product per second每秒钟催化转化1摩尔底物所需的酶量nKatal:mol.s-1Enzyme activityV0Ukat
12、(initial rate)mmol.min-1(enzyme unit)mol.s-1mmol.min-1(SI unit)1mmol min-1=1U=16.67 nanokatExpression of enzyme activity酶活性的表示酶活性的表示 11Some expressions of the components of an enzyme酶的成分的几种表示方式酶的成分的几种表示方式nEnzyme 酶nProtein part+Cofactors(Non-protein part)(蛋白部分+辅助因子(非蛋白部分)nHoloenzyme全酶nApoenzyme+Prost
13、hetic group(脱辅基酶+辅基)Cofactors 辅助因子辅助因子nInorganic nInorganic ion(s)无机离子 Fe2+,Zn2+,Cu2+,etc.nInorganic cluster(s)无机簇 Fe-S cluster,Mn-O complex,etcnOrganic nCoenzyme 辅酶 Which loosely bind to their enzymes nProsthetic group 辅基 Which tightly bind to their enzymesThe important coenzymes and prosthetic gro
14、ups 重要的辅酶和辅基重要的辅酶和辅基nCoenzymes 辅酶nCoenzyme A (CoA)nNAD(H),NADP(H)nNicotinamide adenine dinucleotide 烟酰胺腺嘌呤二核苷酸nNicotinamide adenine dinucleotide phosphatenProsthetic groups 辅基nFAD(H2),FMN(H2)*nFlavin adenine dinucleotide 黄素腺嘌呤二核苷酸nFlavin mononucleotide 黄素单核苷酸nBiotin 生物素nHeme 血红素*FAD and FMN in most
15、cases are prosthetic groups binding to flavin proteinsnWater soluble vitamins(Vitamin B)nThiamine(B1)硫胺素nIn Coenzymes：thiamine pyrophosphate 存在于硫胺焦磷酸辅酶中nRiboflavin(核黄素B2)nAs prosthetic group flavin protein:FAD and FMNnNicotinic acid(niacin烟碱酸)nIn Coenzymes:NAD and NADPnPantothenic acid(泛酸）nIn Coenzy
16、me AnBiotin(生物素）nIn prosthetic group:Biocytin生物胞素nLipid soluble vitamins(Vitamin A,D,E,K)nVitamin AnRetinal 视网膜素：vision,growth and reproductionMost coenzymes are derived from vitamins大多数辅酶源于维生素大多数辅酶源于维生素Some common coenzymes,their vitamin precursors and deficiency diseases Coenzyme/prosthetic groupP
17、recursorDeficiency diseaseCoenzyme APanthothenic acidDermatitis皮肤炎FAD,FMNRiboflavin(Vitamin B2)Growth retardationNAD+(Coenzyme I),NADP+(Coenzyme II)Niacin(菸碱酸)Pellagra 玉蜀黍疹Thiamine pyrophosphateThiamine(Vitamin B1)Beriberi 脚气病TetrahydrofolateFolic acidAnemia贫血症Deoxyadenosyl cobalamin（脱氧腺苷钴胺素）Cobalam
18、in(Vitamin B12)Pernicious anemia恶性贫血症Co-substrate in the hydroxylation of proline in collagenVitamin C(ascorbic acid)Scurvy 坏血病Pyridoxal phosphate 磷酸吡哆醛Pyridoxine(vitamin B6)Dermatitis 皮肤炎Coenzyme A3-phospho-adenosine phosphate3-磷酸ADPHHHSOCNCH2CH2OO-OPOO-OPOCH2CH3CH3OHCCHOCNCH2CH24-phosphopantethein
19、e(4-磷酸泛酰巯基乙胺)-mercaptoethylamine-巯基乙胺Pantothenic acid(泛酸)Ribose 3phosphate核糖3磷酸OHOHOHCH2HHPO-OO-NNNNNH212345Transfer of acetyl groupNAD+,NADP+Coenzyme ofDehydrogenasesNAD(P)+H+2 e-NAD(P)HReductionOxdationNAD+,NADP+H+2 e-H-NAD(P)+H-NAD(P)HReductionOxdationGeneral reactionFAD,FMNFig.18.21,p591Isoallox
20、azine 异咯嗪异咯嗪Flavin mononucleotide,FMNFlavin adenine dinucleotide,FADRiboflavinAMPRibitol核醣醇NNNNH3CH3COORHHH2 H+2 e-NNNNH3CH3CHRHHOHOFAD,FMNFAD+2H+2 e-FADH2ReductionOxdationFMN+2H+2 e-FMNH2ReductionOxdationChemical structure of biotinChapter 14 Enzyme kinetics n Introduction to enzymes 酶（学）导论n Factor
21、s affecting enzyme activity 影响酶活性的因素n Enzyme assaysn Enzyme kinetics and inhibition 酶动力学和抑制作用nCofactors 辅助因子辅助因子(SEE the last section)npHnTemperaturenSubstrate concentration 底物浓度底物浓度nProduct concentrationFactors affecting enzyme activity Chapter 14 EnzymesEffect of pHPepsin胃蛋白酶胃蛋白酶Papain木瓜蛋白酶木瓜蛋白酶Tr
22、ypsin胰蛋白酶胰蛋白酶Cholinesterase胆碱酯酶胆碱酯酶过氧化氢酶Effect of temperaturen50 C,decrease due to denaturation of enzymeBATypicallyEnzyme assays 酶的检测酶的检测nTwo most convenient waysnDetg.the rate of appearance of product 测定产物形成的速率nDetg.the rate of disappearance of substrate 测定反应物减少的速率nA linked enzyme assay way 酶联检测法n
23、First enzyme reaction+second enzyme reactionMeasurements 测定测定1.Spectrophotometry 分光光度法分光光度法 2.Enzyme electrode 酶电极法酶电极法LDH:lactate dehydrogenase 乳酸脱氢酶COO-CHOHCH3+NAD+COO-COCH3Pyruvate丙酮酸Lactate乳酸+H+NADH+A340nmLDHCHOCCCCCH2OHOHOHHOHHHOHHH2O2COOHCCCCCH2OHOHOHHOHHHOHHGlucose oxidase+O2+H2O1/2GlucoseGlu
24、conic acidPeroxidaseH2O2ColorlesscompoundOxidized coloredcompoundH2O葡萄糖氧化酶过氧化氢氧化酶葡萄糖酸Chapter 14 Enzyme kinetics n Introduction to enzymes 酶（学）导论n Factors affecting enzyme activity 影响酶活性的因素n Enzyme assaysn Enzyme kinetics and inhibition 酶动力学和抑制作用Enzyme kinetics and inhibition(酶动力学和抑制作用酶动力学和抑制作用)nCata
25、lytic mechanism 催化机制催化机制 nEnzyme kinetics 酶动力学酶动力学nMichaelis-Menten modelnEnzyme inhibition 酶的抑制酶的抑制nReversible inhibition 可逆抑制可逆抑制nNonreversible inhibition 非可逆抑制非可逆抑制Enzymes catalyze chemical reaction through lowering its activation energyG0:not thermodynamically favorable Progress of reactionFree
26、energy,GTransition state(uncatalyzed)Transition state(catalyzed)GuncatalyzedGcatalyzedSPGS:substrates 底物P:productsGGGibbs free energy of activation吉布斯活化自由能Gibbs free energy change吉布斯自由能变化Activation Energy Rate Constant of Reaction Gk nArrhenius equationk=Ae-G/RTGknQuestions(self study)nWhat does dyn
27、amic equilibrium mean?nEnzymes can accelerate the attainment of the equilibrium position,can it change this position?nWhats role of active site of an enzyme?nBy which forces the substrate binds to the active site of an enzyme?Chemical equilibrium:(self study)Michaelis-Menten modelE +SESE +Pk1k2k3max
28、0SKSVVm132kkkKmVmax/2VmaxV0SKmHyperbolic curve双曲线111maxmax0SVKVVmDouble reciprocal or lineweaver-Burk plot1/S1/V0Intercept=1/VmaxSlope=Km/VmaxIntercept=-1/KmV0Time(min)Amount of product formed(mmol)Measurement and calculation of V0Enzyme inhibition 酶的抑制nInhibition 抑制的定义nA phenomenon of lowering enzy
29、mes catalytic rate.nInhibitor 抑制剂nAny molecule which acts directly on an enzyme to lower its catalytic ratenTwo main types of inhibitionsnIrreversible 非可逆抑制(抑制)nReversible 可逆(抑制)nCompetitive 竞争性nNoncompetitive 非竞争性nMixed noncompetitive inhibition 混合型非竞争性Irreversible inhibition by covalent binding be
30、tween I and E nResidues to be boundnSusceptible amino acid residues near the active sitenSer,with OH groupnCys,with SH group nPrinciplenChange molecular conformation or inhibit approaching of the substrate to the active site of enzyme I +E IECovalent+SI:inhibitorE:enzymeS:substrateiodoacetamide 碘乙酰胺
31、+(CH3)2CHOPFOCH(CH3)2O(CH3)2CHOPOCH(CH3)2OOEnzymeCH2HFEnzymeCH2SH+ICH2CNH2OEnzymeCH2SCH2CNH2OHIEnzymeCH2OHDiisopropylphospho-fluoridate(DIPF)二异丙基磷酰氟 ExamplesReversible competitive inhibition nBoth I and S compete with E at the active site 活性位点活性位点.nI binds(noncovalently)only to E I,S:closely similar
32、 in structureESESE +PEIII,S:closely similar in structureReversible noncompetitive inhibition nThe inhibitor binds reversibly at a site other than the active site（非活性位点）（非活性位点）nI binds noncovalently to both E and ES 1/V01/SNo inhibitor+Compettitive inhibitor1/Vmax-1/KmESESE +PEIIESIISMixed noncompetitive inhibitionI binds at a site distinct from active site,yet affect thebinding of S at the active siteHomeworkProblems 1-7Pp458-459
