生物化学考研课件ch14酶动力学enzymekinetics

1、BiochemistryFor Speciality of Biotechnology or Bioengineering at CSUMr. XIA JinlanChapter 14 Enzyme kinetics Introduction to enzymes 酶（学）导论Factors affecting enzyme activity 影响酶活性的因素Enzyme assaysEnzyme kinetics and inhibition 酶动力学和抑制作用Introduction to enzymes 酶（学）导论The features of enzymes 酶的特征Some ter

2、ms of enzyme 酶的几种术语Classes of enzymes 酶的种类Enzyme Nomenclature 酶的命名法Traditional nomenclature 传统命名法Systematic nomenclature 系统命名法Expression of enzyme activity 酶活性的表示Components of enzyme 酶的成分Cofactors 辅助因子Powerful Catalytic power 强的催化力Increase reaction rate up to 1016 foldMake reaction taken place under

3、 relatively mild conditions of temperature and pHHigh specificity 高的特异性To the substrates that they act on and the products they formEnzyme activity can be regulated 酶活性可以被调控Basically are proteins, except few ribozymes 除了少量的核酶外,基本上是蛋白质 Enzymes have four basic features酶的四个基本特征Some terms of enzymes 酶的几

4、种术语Proteoenzyme蛋白酶Ribozyme核酶Abzyme 抗体酶Holoenzyme全酶Apoenzyme脱辅酶Multimeric 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,酶可分为六种类型Oxidoreductases 氧化还原酶Transfer of electrons 电子的转移Transferases 转化(或转移)酶Transfer of functional groups 分子间功能基团的转移Hydr

5、olases 水解酶Hydrolysis reactions 水解反应Lyases 裂解酶Cleavage 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Isomerases 异构酶Transfer of groups within a molecule 分子内基团的转移Ligases (or synthases) 链接酶(或合成酶)Bond formation coupled to ATP hydrolysis伴随ATP水解的键合Enzymes are classified int

6、o six classes based on the type of reaction catalyzed NameType of reaction catalyzedExampleOxido-reductasesTransfer of electronsA- + B A + B-Alcohol dehydrogenaseTransfer-asesTransfer of functional groupsA-B +CA + B-CHexokinaseHydrolasesHydrolysis reactionsA-B + H2OA-H + B-OHTrypsinLyasesCleavage of

7、 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 carboxylaseA + B A-BX-A-B-Y Y-A-B-XX-A-B-Y A=B + Y-XTraditional Nomenclature of enzymesSystematic

8、nomenclature: Each enzyme is uniquely identified by a four-digit classification number Enzyme commission (EC) number: Four-digit classification numberWhat do these numbers signify? Trypsin EC 3.4.21.4Chymotrypsin EC 3.4.21.1Elastase EC 3.4.21.36Expression of enzyme activity 酶活性的表示 Initial rate (V0)

9、of the reaction mmol of substrate transformed per minute 每分钟转化底物的微摩尔数V0: X mmol.min-1 Enzyme unit 酶的单位, SI unitThe 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U: mmol.min-1Katal , SI unitAmount of enzyme catalyzing the conversion of one mole of substrate

10、 to product per second每秒钟催化转化1摩尔底物所需的酶量Katal: mol.s-1Expression of enzyme activity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Enzyme 酶Protein part + Cofactors (Non-protein part) (蛋白部分+辅助因子(非蛋白部分) Holoenzyme全酶Apoenzyme + Prosthetic group (脱辅基酶+辅基)Cofactors 辅助因子Inorganic Inorgan

11、ic ion(s) 无机离子 Fe2+, Zn2+, Cu2+, etc.Inorganic cluster(s) 无机簇 Fe-S cluster, Mn-O complex,etcOrganic Coenzyme 辅酶 Which loosely bind to their enzymes Prosthetic group 辅基 Which tightly bind to their enzymesThe important coenzymes and prosthetic groups 重要的辅酶和辅基Coenzymes 辅酶Coenzyme A (CoA)NAD(H), NADP(H)

12、Nicotinamide adenine dinucleotide 烟酰胺腺嘌呤二核苷酸Nicotinamide adenine dinucleotide phosphateProsthetic groups 辅基FAD(H2), FMN(H2)*Flavin adenine dinucleotide 黄素腺嘌呤二核苷酸Flavin mononucleotide 黄素单核苷酸Biotin 生物素Heme 血红素* FAD and FMN in most cases are prosthetic groups binding to flavin proteinsWater soluble vit

13、amins (Vitamin B)Thiamine (B1) 硫胺素In Coenzymes：thiamine pyrophosphate 存在于硫胺焦磷酸辅酶中Riboflavin (核黄素B2)As prosthetic group flavin protein: FAD and FMNNicotinic acid (niacin烟碱酸)In Coenzymes: NAD and NADPPantothenic acid (泛酸）In Coenzyme ABiotin (生物素）In prosthetic group: Biocytin生物胞素Lipid soluble vitamins

14、(Vitamin A, D, E, K)Vitamin ARetinal 视网膜素：vision, growth and reproductionMost coenzymes are derived from vitamins大多数辅酶源于维生素Some common coenzymes, their vitamin precursors and deficiency diseases Coenzyme/prosthetic groupPrecursorDeficiency diseaseCoenzyme APanthothenic acidDermatitis皮肤炎FAD, FMNRibof

15、lavin (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）Cobalamin (Vitamin B12)Pernicious anemia恶性贫血症Co-substrate in the hydroxylation

16、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 ATransfer of acetyl groupNAD+, NADP+Coenzyme ofDehydrogenasesNAD(P)+ + H+ + 2 e-NAD(P)HReductionOxdationNAD+, NADP+H+ + 2 e-H-NAD(P)+ + H-NAD(P)HReductionOxdationGeneral re

17、actionFAD, FMNFig. 18.21, p591Isoalloxazine 异咯嗪Flavin mononucleotide, FMNFlavin adenine dinucleotide, FADRiboflavinAMPRibitol核醣醇FAD, FMNFAD + 2H+ + 2 e-FADH2ReductionOxdationFMN + 2H+ + 2 e-FMNH2ReductionOxdationChemical structure of biotinChapter 14 Enzyme kinetics Introduction to enzymes 酶（学）导论Fac

18、tors affecting enzyme activity 影响酶活性的因素Enzyme assaysEnzyme kinetics and inhibition 酶动力学和抑制作用Cofactors 辅助因子 (SEE the last section) pHTemperatureSubstrate concentration 底物浓度Product concentrationFactors affecting enzyme activity Chapter 14 EnzymesEffect of pHPepsin胃蛋白酶Papain木瓜蛋白酶Trypsin胰蛋白酶Cholinestera

19、se胆碱酯酶过氧化氢酶Effect of temperature50 C, decrease due to denaturation of enzymeBATypicallyEnzyme assays 酶的检测Two most convenient waysDetg. the rate of appearance of product 测定产物形成的速率Detg. the rate of disappearance of substrate 测定反应物减少的速率A linked enzyme assay way 酶联检测法First enzyme reaction + second enzym

20、e reactionMeasurements 测定Spectrophotometry 分光光度法 Enzyme electrode 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+A340nmLDH葡萄糖氧化酶过氧化氢氧化酶葡萄糖酸Chapter 14 Enzyme kinetics Introduction to enzymes 酶（学）导论Factors affecting enzyme activity 影响酶活性的因素Enzyme assaysEnzyme

21、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)Catalytic mechanism 催化机制 Enzyme kinetics 酶动力学Michaelis-Menten modelEnzyme inhibition 酶的抑制Reversible inhibition 可逆抑制Nonreversible inhibition 非可逆抑制Enzymes catalyze chemical reaction through lowering its activation energyG0: not

22、 thermodynamically favorable Progress of reactionFree energy, GTransition state(uncatalyzed)Transition state(catalyzed)GuncatalyzedGcatalyzedSPGActivation Energy Rate Constant of Reaction Gk Arrhenius equationk =Ae-G/RTGkQuestions (self study) What does dynamic equilibrium mean?Enzymes can accelerat

23、e the attainment of the equilibrium position, can it change this position?Whats role of active site of an enzyme? By which forces the substrate binds to the active site of an enzyme?Chemical equilibrium: (self study)Michaelis-Menten modelHyperbolic curve双曲线Double reciprocal or lineweaver-Burk plotV0

24、Time (min)Amount of product formed (mmol)Measurement and calculation of V0Enzyme inhibition 酶的抑制Inhibition 抑制的定义A phenomenon of lowering enzymes catalytic rate. Inhibitor 抑制剂Any molecule which acts directly on an enzyme to lower its catalytic rateTwo main types of inhibitionsIrreversible 非可逆抑制(抑制)Reversible 可逆(抑制)Competitive 竞争性Noncompetitive 非竞争性Mixed noncompetitive inhibition 混合型非竞争性Irreversible inhibition by covalent bin