Chapter 3. enzyme Composition Characteristics and mechanism Kinetics of enzyme-catalzed reaction Regulation Names and classes Enzyme and medicine 2018/11/21

introduction biocatalyst生物催化剂: enzyme and ribozyme核酶 Enzyme---proteins having catalytic activity Ribozyme---nucleic acids having catalytic activity 2018/11/21

Enzyme is protein. 1926, Sumner美国,脲酶结晶，酶是蛋白质 J.B.Sumner J.H.Northrop 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，进一步证明了酶是蛋白质 2018/11/21

ribozyme 1982年T.Cech等人发现四膜虫的rRNA前体能在完全没有蛋白质的情况下进行自我加工，发现RNA有催化活性 Thomas Cech University of Colorado at Boulder, USA 1982年T.Cech等人发现四膜虫的rRNA前体能在完全没有蛋白质的情况下进行自我加工，发现RNA有催化活性 1983年S.Altman等研究RNaseP（20%蛋白质和80%RNA），发现RNaseP中的RNA可催化E. coli tRNA的前体加工 Sidney Altman Yale University New Haven, CT, USA 2018/11/21

Section 1 molecular structure and function Monomeric enzyme单体酶：one peptide chain Oligomeric enzyme寡聚酶：many subunits Multienzyme system多酶体系：complex of enzymes of different function. Multifunctional enzyme多功能酶 / Tandem enzyme串联酶: function different enzymes in one peptide chain. 2018/11/21

Ⅰ. Molecular composition of enzymes inactive Simple enzyme单纯酶 enzyme apoenzyme酶蛋白 metal ion金属离子 Conjugated enzyme结合酶 cofactor辅助因子 Holoenzyme 全酶 active Small organic molecules　有机小分子 2018/11/21

Functions of metal ion: Metalloenzyme金属酶:　bind metal ion tightly Metal activated enzyme金属激活酶: don’t bind metal ion tightly, but the metal ion is essential to the enzyme activity. Functions of metal ion: As active center, join in electron transfer,group tansfer and so on. Stablize steric structure of apoenzyme. Help the substrate binding. Neutralize anion, to lower static electricity repulsion of the reaction. 2018/11/21

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coenzyme辅酶 cofactor Prosthetic group辅基 2018/11/21

Ⅱ. Active center of enzymes 2018/11/21

Section 2. characteristics and mechanism of enzyme-catalyzed reaction 酶与一般催化剂的共同点common characters of enzymes and general catalysts： 反应前后没有质和量的变化the quality and mass of catalysts before the reaction are identical to that after reaction. 只能催化热力学允许的反应catalyze the reaction thermodynamics-permissive only. 只能加速可逆反应的进程，而不能改变反应的平衡点Accelerate reversible reaction proceeding, but can’t change the point of balance. 2018/11/21

Ⅰ. Characteristics of enzyme-catalyzed reaction High efficiency 比非催化反应高108～1020倍； 可比一般催化剂高106～1013倍 尿素水解 脲酶／Ｈ＋：７１０１２ 苯酰胺水解 α-胰凝乳蛋白酶／Ｈ＋：6 106 25℃时，活化能每减少4.18kJ/mol,反应速度增加5.4倍． 2018/11/21

The enzyme-catalyzed reaction can be regulated可调节性 Specificity专一性 Absolute specificity: 一种底物 Relative specificity：一类底物 Stereospecificity：对立体异构体有选择性 The enzyme-catalyzed reaction can be regulated可调节性 2018/11/21

Ⅱ. Mechanism of enzyme-catalyzed reaction Induced-fit hypothesis for forming of enzyme-substrate complex 　　酶在与底物相互接近时，其结构相互诱导、相互变形和相互适应，进而相互结合。 2018/11/21

Reaction mechanism 邻近效应proximity effect与定向排列orientation arrange 多元催化multielement catalysis与协同作用cooperation 表面效应surface effect 2018/11/21

Section 3. kinetics of enzyme-catalyzed reactions Ⅰ. Influence of substrate concentration on reaction velocity 当底物浓度很小时，为一级反应．V= k [S] 随底物浓度逐渐增大，变为混合级反应 当底物浓度很大时，为零级反应.v= k. 2018/11/21

1. Michaelis equation米－曼氏方程式 1913, L. Michaelis and M.L.Menten 2018/11/21

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Km equal to [S] when the velocity is half of the Vmax. (mol/L) 2. Meaning of Km and Vm Km equal to [S] when the velocity is half of the Vmax. (mol/L) Km的大小反映该酶对底物亲和力的大小the value of Km weigh the affinity of enzyme with substrate. Km= K2+K3 K1 Km= K2 K1 = KS = [E] [S] [ES] If K2 >> K3, Km值是酶的特性常数，只与酶的结构、底物和反应环境有关，与酶的浓度无关。在10-6~10-2mmol/L之间。 2018/11/21

Vm, Vmax, 最大反应速度，酶完全被底物饱和时的反应速度． K3, 转换数 turnover number: 　　当酶被底物充分饱和时，单位时间内每个酶分子催化底物转变为产物的分子数．大多数在1~104/s之间． K3 = Vm [E] 2018/11/21

3. Determination of Km and Vm 1 V = Km Vm [S]  + 1 V Km Vm r = 1 Vm 1 Km - 1 [S] 2018/11/21

Ⅱ. influence of enzyme concentration v = k3 [E], as [S]>>[E] 2018/11/21

Ⅲ. Influence of temperature Optimum temperature最适温度，most of them are in the range from 35 to 45℃ . 2018/11/21

Ⅳ. Influence of pH Optimum pH最适pH 常为中性，胃蛋白酶为1.8, 肝精氨酸酶为9.8． 2018/11/21

Ⅴ. Influence of inhibitors the matters which decrease enzyme activity, but don’t make the enzyme denatured. Irreversible inhibition Reversible inhibition可逆性抑制 competitive inhibition竞争性抑制 non-competitive inhibition非竞争性抑制 uncompetitive inhibition反竞争性抑制 2018/11/21

1. Irreversible inhibition the inhibitor combine with essential group of enzyme active center by covalent bond, resulting in enzymatic activity loss. 2018/11/21

2. Reversible inhibition the inhibitor combine with enzyme,or enzyme-substrate complex by non-covalent bond, resulting in enzymatic activity decrease or loss. the inhibitor can be removed by dialysis, or ultrafiltration competitive inhibition竞争性抑制 non-competitive inhibition非竞争性抑制 uncompetitive inhibition反竞争性抑制 2018/11/21

(1) competitive inhibition the structure of inhibitor is similar to substrate. This make it combine with enzyme competively to substrate. 2018/11/21

Characteristics of competitive inhibition Inhibitor structure is similar to that of substrate Degree of inhibition is determined by their affinity and substrate concentration. Vmax is invariable, apparent Km increase. 2018/11/21

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(2)non-competitive inhibition非竞争性抑制 2018/11/21

Characteristics of non-competitive inhibition Inhibitor combine with essential groups out of active center, no competitive . Degree of inhibition is determined by the concentration of inhibitor. Vmax decrease, Km is invariable 2018/11/21

(3)uncompetitive inhibition反竞争性抑制 2018/11/21

Characteristics of uncompetitive inhibition Inhibitor combine with enzyme-substrate complex only. Degree of inhibition is determined by concentration of both inhibitor and substrate. Vm decrease, Km decrease. 2018/11/21

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Ⅵ. Influence of activator Activator激活剂：substance which change enzymes inactive into active state, or which increase enzyme activity, such as Mg2+, K+, Cl-. Essential activator Non-essential activator 2018/11/21

Ⅶ. Determination of enzyme activity ; enzyme activity unit The enzyme activity is determined by the reaction ratio. 1国际单位 IU = 在特定条件下，每分钟催化1μmol底物转化为产物所需要的酶量．Quantity of enzyme which can catalyze 1μmol substrate change into product at certain condition. 1催量kat＝在特定条件下，每秒钟催化1mol底物转化为产物所需要的酶量． 1IU = 16.67  10-9 kat 2018/11/21

Section 4. the regulation of enzyme Regulated target: key enzyme Ways of regulation: Regulation of enzyme activity (fast). Regulation of enzyme quantity (slow). 2018/11/21

Ⅰ. Regulation of enzyme activity 1.Zymogen and activation of zymogen酶原与酶原激活 Zymogen: 有些酶在细胞内合成或刚分泌时，无催化活性，这种无催化活性的酶的前体． activation of zymogen： the process of inactive zymogen change into active enzyme, also the process of forming or exposing active center. 2018/11/21

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2. Allosteric regulation变构调节 效应剂 2. Allosteric regulation变构调节 调节亚基 催化亚基 变构酶 2018/11/21

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3. Regulated by covalent modification of enzyme 磷酸化phosphorylation  脱磷酸化dephosphorylation 乙酰化acetylation  脱乙酰化产deacetylation 甲基化methylation  脱甲基化demethylation 腺苷化adenosination  脱腺苷化deadenosination RSH  RS-SR 2018/11/21

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Ⅱ. Regulation of enzyme quantity Induce or repress synthesis of apoenzyme Regulation of enzyme degradation 2018/11/21

Ⅲ. Isoenzyme同工酶 Isoenzyme: enzymes which catalyze same reaction, but their molecule structure, physico-chemical property　and immunology charcter are different.they must be the enzymes encoded by different genes or mRNA, exist in different tissues of a body,or in different subunits of a cell. 2018/11/21

Section 5. naming and classification of enzyme Ⅰ. 命名 习惯名称推荐名称 系统命名法： 底物：底物　反应性质． 2018/11/21

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Ⅱ. classification 氧化还原酶oxidoreductases 转移酶transferases 水解酶hydrolases 裂解酶lyases：催化从底物移去一个基团并留下双键的的反应或其逆反应． 异构酶isomerases 合成酶ligases 2018/11/21

Section 6. relation of enzyme with medicine Ⅰ. Enzyme and disease Enzyme and diseases occurrence Enzyme and diseases diagnosis Enzyme and diseases treatment 2018/11/21

Ⅱ. Application of enzyme in medicine used in clinical test as reagent Used in clinical treatment as medicine Used in scientific research and production 工具酶 　酶标记测定法 　固定化酶 　抗体酶 2018/11/21

选择题练习 酶化学 2018/11/21

E．酶是由活细胞合成具有催化作用的蛋白质 1. 关于酶概念的叙述下列哪项是正确的？ A．所有蛋白质都有酶的活性 B．其底物都是有机化合物 C．其催化活性都需要特异的辅助因子 D．体内所有具有催化活性的物质都是酶 E．酶是由活细胞合成具有催化作用的蛋白质 2018/11/21

2．关于酶活性中心的叙述下列哪项是正确的？ A．所有酶的活性中心都有金属离子 B．所有的抑制剂都作用于酶的活性中心 C．所有的必需集团都位于酶的活性中心 D．所有酶的活性中心都含有辅酶 E．所有的酶都有活性中心 2018/11/21

3. 酶加速化学反应的根本原因是( ) A. 升高反应温度 B. 增加反应物碰撞频率 C. 降低催化反应的活化能 D. 增加底物浓度 3. 酶加速化学反应的根本原因是( ) A. 升高反应温度 B. 增加反应物碰撞频率 C. 降低催化反应的活化能 D. 增加底物浓度 E. 降低产物的自由能 2018/11/21

A. Complex of enzyme with substrate 4. Holoenzyme refer to ( ) A. Complex of enzyme with substrate B. Complex of enzyme with suppressant C. Complex of enzyme with cofactor D. Inactive precursor of enzyme Complex of enzyme with allosteric effector 2018/11/21

5. 金属离子作为辅助因子的作用错误的是( ) A. 作为酶活性中心的催化基团参加反应 B. 与稳定酶的分子构象无关 5. 金属离子作为辅助因子的作用错误的是( ) A. 作为酶活性中心的催化基团参加反应 B. 与稳定酶的分子构象无关 C. 可提高酶的催化活性 D. 降低反应中的静电排斥 E. 可与酶、底物形成复合物 2018/11/21

6. 活化能的概念是指( ) A. 底物和产物之间能量的差值 B. 参与反应的分子所需的总能量 C. 分子由一般状态变成活化态所需能量 6. 活化能的概念是指( ) A. 底物和产物之间能量的差值 B. 参与反应的分子所需的总能量 C. 分子由一般状态变成活化态所需能量 D. 温度升高时产生的能量 E. 以上都不是 2018/11/21

7. 酶促反应动力学所研究的是( ) A. 酶的基因来源 B. 酶的电泳行为 C. 酶的诱导契合 D. 酶分子的空间结构 7. 酶促反应动力学所研究的是( ) A. 酶的基因来源 B. 酶的电泳行为 C. 酶的诱导契合 D. 酶分子的空间结构 E. 影响酶促反应速度的因素 2018/11/21

8. Michaelis-Menten enzyme kinetics diagram of curves is a ( ) A. straight line B. rectangular hyperbola C. S shape curve D. parabola E. Not above all 2018/11/21

9. 关于Km的意义正确的是( ) A. Km为酶的比活性 B. 1/Km越小,酶与底物亲和力越大 C. Km的单位是mmol/min D. Km值是酶的特征性常数之一 E. Km值与酶的浓度有关 2018/11/21

10. 竞争性抑制剂的特点是( ) A. 抑制剂以共价键与酶结合 B. 抑制剂的结构与底物不相似 C. 当抑制剂的浓度增加时,酶变性失活 10. 竞争性抑制剂的特点是( ) A. 抑制剂以共价键与酶结合 B. 抑制剂的结构与底物不相似 C. 当抑制剂的浓度增加时,酶变性失活 D. 当底物浓度增加时,抑制作用不减弱 E. 抑制剂和酶活性中心外的部位结合 2018/11/21

11. In anticompetitive inhibition of enzyme, the reaction kinetics parameter change as ( ) Km↑,Vmax invariably Km↓,Vmax↓ Km invariably,Vmax↓ D. Km↓,Vmax invariably E. Km↓,Vmax↑ 2018/11/21

12. 有机磷农药与酶活性中心结合的基团是( ) A. 组氨酸上的咪唑基 B. 赖氨酸上的ε-氨基 C. 丝氨酸上的羟基 12. 有机磷农药与酶活性中心结合的基团是( ) A. 组氨酸上的咪唑基 B. 赖氨酸上的ε-氨基 C. 丝氨酸上的羟基 D. 半胱氨酸上的巯基 E. 谷氨酸上的γ-羧基 2018/11/21

B. 如底物与一亚基结合后,使其他亚基迅速与底物结合程正协同效应 13. 关于变构酶的论述错误的是( ) A. 变构酶为多亚基组成 B. 如底物与一亚基结合后,使其他亚基迅速与底物结合程正协同效应 C. 正协同效应的底物浓度曲线呈矩形双曲线 D. 底物与一亚基结合后,使其亚基结合底物能力减少称负协同效应 E. 变构效应剂与一亚基结合后,使酶其他亚基迅速与底物结合为异促协同效应 2018/11/21

14. –SH is one enzyme’s essential group 14. –SH is one enzyme’s essential group. Which substance can protect this enzyme from oxidation? A. Cys B. GSH C. urea D. ionic detergent E. ethanol 2018/11/21

15. 快速调节可通过( ) A. 磷酸化与去磷酸化 B. 腺苷酸化与腺苷酸化 C. 变构调节 D. 改变酶的合成速度 15. 快速调节可通过( ) A. 磷酸化与去磷酸化 B. 腺苷酸化与腺苷酸化 C. 变构调节 D. 改变酶的合成速度 E. 酶促反应的可调节性 2018/11/21

16. The characteristic constants of enzymes include ( ) A. Enzymic optimum temperature B. Enzymic optimum pH C. Vmax D. Km E. KS 2018/11/21

D. 抑制程度强弱取决于药物与酶底物浓度的相对比例 17. 磺胺药的抑菌机理是( ) A. 竞争性抑制二氢叶酸合成酶的活性 B. 干扰体内核酸的代谢 C. 结构与二氢叶酸相似 D. 抑制程度强弱取决于药物与酶底物浓度的相对比例 E. 磺胺药是二氢叶酸合成酶的变构抑制剂 2018/11/21

18. Cofactors of enzyme are ( ) A. Micromolecule organic compounds B. metal ion C. vitamine D. various kinds of organic and inorganic compounds E. A kind of conjugated protein 2018/11/21

19. 某种酶的活性依赖于酶活性中心的必需基团-SH,能保护此酶不被氧化的物质是( ) A. GSH B. 维生素C C. 半胱氨酸 D. 维生素A E. 两价阳离子 2018/11/21

20. 酶分子上必需基团的作用是( ) A. 与底物结合 B. 催化底物发生化学反应 C. 含砷的有机化合物 D. 决定辅酶结构 20. 酶分子上必需基团的作用是( ) A. 与底物结合 B. 催化底物发生化学反应 C. 含砷的有机化合物 D. 决定辅酶结构 E. 维持酶分子空间结构 2018/11/21

Thank you! 2018/11/21