第三章 酶( enzyme) - PowerPoint PPT Presentation

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第三章 酶( enzyme)

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  1. 第三章酶(enzyme) 主要介绍酶的化学本质、结构和特性;酶的作用动力学;酶的作用机理;酶的应用;还介绍了别构酶、共价调节酶、同工酶等的概念、性质、生物学意义。 主讲老师:华南师范大学生命科学学院      陈文利

  2. ★ Study history, ★ General features, ★ Classification, ★ Chemical nature, ★ Kinetics ★ Mechanism, ★ Regulation

  3. 酶通论 • 酶的发现和提出:1897年,Buchner兄弟用不含细胞的酵母汁成功实现了发酵。提出了发酵与活细胞无关,而与细胞液中的酶有关。 • 1903年,Henri提出了酶与底物作用的中间复合物学说。 • 1913年,Michaelis和Menten提出了酶促动力学原理—米氏学说。 • 1925年,Briggs和Handane对米氏方程做了修正,提出了稳态学说。 • 1926年,Sumner从刀豆种子中分离、纯化得到了脲酶结晶,首次证明酶是具有催化活性的蛋白质。 • 1930年 Northrop 分离得到胃蛋白酶、胰蛋白酶和胰凝乳蛋白酶结晶并证实其均为蛋白质,酶的蛋白质本质确立。

  4. Urease crystals ( X 728)Sumner, J. B. (1926) “ The isolation and crystallization of the enzyme urease”J. Biol. Chem. 69:435-441.

  5. Pepsin crystals (X90) Northrop, 1930) Northrop, J. H. (1930) “Crystallin pepsin, 1: Isolation and tests of purity”J. Gen . Physiol. 13:739-766.

  6. 1969年,Merrifield等人工合成了具有酶活性的胰RNase。1969年,Merrifield等人工合成了具有酶活性的胰RNase。 1982年,Cech和Altman对四膜虫的研究中发现RNA具有催化作用,从而发现核酶(ribozyme),打破了以往酶是蛋白质的传统观念。 1986年Schultz和Lerner等人研制成功抗体酶(abzyme)。 Boyer和Walker阐明了ATP合酶的合成与分解ATP的分子机制。 近20年来,不少酶的作用机制被阐明,随着基因工程技术的广泛应用,使得酶的结构和功能的研究进入一个新的阶段。现在已鉴定出4000多种酶,数百种酶已经被结晶。 近几十年来,不断有新理论和新概念出现。在分子水平上揭示酶和生命活动的关系,阐明酶在细胞代谢调节和分化中的作用,酶合成的遗传机制,酶的催化机制等。 同时,酶的应用——酶工程也得到快速发展。

  7. 第一节 通论 一、酶是生物催化剂 酶的定义:P110 酶是由活细胞产生的,能在体内或体外起同样催化作用的一类具有活性中心和特殊构象的生物大分子,包括蛋白质和核酸。

  8. 酶的化学本质 1.大多数酶是蛋白质(Most enzymes are proteins) 1926年美国Sumner 脲酶的结晶,并指出酶是蛋白质 1930年Northrop等得到了胃蛋白酶、胰蛋白酶和胰凝乳蛋白酶的结晶,并进一步证明了酶是蛋白质。 J.H.Northrop J.B.Sumner

  9. 20世纪80年代发现某些RNA有催化活性,还有一些抗体也有催化活性,甚至有些DNA也有催化活性,使酶是蛋白质的传统概念受到很大冲击。

  10. What is the difference between an enzyme and a protein? Protein RNA Enzymes All enzymes are proteins except some RNAs and not all proteins are enzymes

  11. Ribozymes • It was assumed that all enzymes are proteins until 1982 when Thomas Cech and Sydney Altman discovered catalytic RNAs (Nobel, 1989 in Chemistry); • Catalytic RNA, or, ribozymes, satisfy several enzymatic criteria: substrate specificity, enhance reaction rate, and emerge from reaction unchanged; • Several known ribozymes: • RNase P: catalyzes cleavage of precursor tRNA molecules into mature tRNAs; • Group I, II introns: catalyze their own splicing (cleaving and ligating); • Ribosome: catalyzes peptidyl transfer reaction

  12. Enzyme Product Substrates Substrates, products, and enzymes Enzymes catalyze the rate at which substrates are converted to product

  13. 二、酶催化的特征 1.高效性 2.易变性 3.可调性 4.专一性(特异性)

  14. Enzymes are the most remarkable and specialized biological catalysts • An enzyme catalyzes a chemical reaction at a specifically structured active site, being often a pocket. • Enzymes have extraordinary catalytic power, often far greater than those non-biological catalysts. • Enzymes often have a high degree of specificity for their substrates. • Enzymes are often regulatory. • Enzymes usually work under very mild conditions of temperature and pH. • The substance acted on by an enzyme is called a substrate, which binds to the active site of an enzyme in a complementary manner.

  15. 降低反应所需的活化能

  16. . . Enzymes catalyze reactions by lowering their activation energies Uncatalyzed Enzymes stabilize the transition states of reactions ) Reaction DG Enzyme Catalyzed Reaction Free Energy ( Substrates D G rxn Products Reaction Coordinate

  17. 例 2H2O2→2H2O+O2

  18. 对酶作用专一性的几个假说P72 锁钥模式(lock and key theory) 三点附着学说 (three point attachment theory) 诱导契合学说(induced-fit theory)

  19. Emil Ficher: (1894) • The ‘Lock & Key’ hypothesis • - explains substrate specificity • - says nothing about why catalysis occurs • Dan Koshland: (1958) • ‘Induced Fit’ hypothesis: • - enzymes prefer to bind to a distortion of the substrate that resembles the transition state • - both enzyme and substrate must adjust to one another • - in reality, enzyme is not ‘distorted’ but has evolved to bind in a certain way and sometimes undergo conformational changes

  20. Substrate + Enzyme ES complex Substrate + ES complex Enzyme Lock and Key - Emil Fischer (1890) Induced Fit - Daniel E. Koshland Jr. (1958)

  21. An Example: Induced conformational change in hexokinase Catalyzes phosphorylation of glucose to glucose 6-phosphate during glycolysis such a large change in a protein’s conformation is not unusual BUT: not all enzymes undergo such large changes in conformation

  22. 1、锁钥学说(lock and Key theory) 1894年 Fischer 提出

  23. 2、三点附着学说

  24. 3、诱导契合学说(induced-fit theory) 1958年 Koshland提出

  25. 第二节 酶的分类和命名 1961年国际酶学委员会(enzyme commission)提出的酶的命名和分类方法。

  26. 和分类

  27. Each enzyme is assigned a systematic name and a four-digit number • The systematic name identifies the substrates and type of reaction catalyzed. • The four-digit number indicates the following: • Class (1-6); • Sub-classes based on type of substrate; • Sub-sub classes based on cofactor type; • A unique number for each individual enzyme within a sub-sub-class.

  28. Each enzyme is given a systematic name and a unique 4-digit identification number for identification lactate + NAD+ pyruvate + NADH + H+ Lactate dehydrogenase (lactate:NAD+ oxidoreductase) 1-6 The number was assigned by the Enzyme Commision (EC) of IUBMB (since 1964).

  29. Enzyme classificationEnzymes are grouped intosixclassesaccording to the type of reactions catalyzed

  30. Lactate dehydrogenase Transfer electrons (hydride ions or H atoms); play a major role in energy metabolism. e.g., the transfer of a phosphoryl group from ATP to many different acceptors. NMP kinase Chymotrypsin i.e., the transfer of functional groups to water. These are direct bond breaking reactions without being attacked by another reactant such as H2O. Fumarase Triose phosphate isomerase In chemical terms, they would be described as elimination and addition reactions. Leading to the formation of C-C, C-S, C-O, C-N bonds. Aminoacyl-tRNA synthetase

  31. 三、酶的组成分类

  32. 酶的组成 1.单纯蛋白质酶类 属于单纯蛋白质 ,主要是水解酶。 2.结合蛋白质酶类 全酶=酶蛋白+辅助因子(辅酶、辅基或金属离子) 辅酶(coenzyme) 辅基(prosthetic group)

  33. 第三节 酶催化作用的结构基础 一、酶分子结构的特征 1、酶的活性部位 酶的活性中心active center (又叫活性部位active site) 定义:在酶分子中,由少数几个氨基酸残基 构成的,直接与底物结合的并和酶的催化作 用直接有关的区域。 通常活性中心有两个功能部位:结合部位 催化部位

  34. 与底物结合的部位叫结合部位(结合基团) binding site 底物分子中的化学键在此处被打断或形成新的化学键叫催化部位(催化基团) catalytic site

  35. (二)酶活性中心的结构特点 活性中心只占酶分子总体积的很小一部分,往往只占整个酶分子体积的1%-2%。 某些酶活性部位的AA残基 酶 AA残基数 活性部位的AA残基 核糖核酸酶 124 His12, His119, Lys41 溶菌酶 129 Asp52, Glu35 胰凝乳蛋白酶 241 His57, Asp102, Ser195 胃蛋白酶 348 Asp32, Asp215 木瓜蛋白酶 212 Cys25, His159 羧肽酶A 307 Arg127, Glu270,Tyr248,Zn2+

  36. Each enzyme has at least one active site

  37. How do enzymes work? Enzymes bind substrates to their active site and stabilize the transition state of the reaction.

  38. What is the active site? The active site of an enzyme is the place where all of the action occurs. It contains the functional groups (amino acid side chains) that bind the substrate(s) and catalyze it’s conversion to product(s).

  39. The active site of the enzyme contains functional groups that stabilize the transition state of the reaction Active siteof chymotrypsin

  40. What does the active site do? • The active site binds the substrates and positions them in the proper orientation for the reaction to occur. • The active site contains chemical groups that stabilize the transition state of the reaction. • The active site determines the specificity of the enzyme (i.e. it determines whether a particular substrate is bound and whether a particular product is made).

  41. Characteristics of active sites • The active site takes up a small part of the total volume of the enzyme • The active site is 3-dimensional and is generally found in a crevice or cleft on the enzyme • The active site displays highly specific substrate binding • The active site is responsible for whether there is ordered or random binding of substrates and release of products