Chapter 3 Enzymes

1. Chapter 3 Enzymes Dept. of biochemistry and molecular biology Hui-Qing Yuan

2. Key terms*** • Active site of enzymes (酶的活性中心) • Allosteric enzymes and allosteric regulation of enzymes (变构酶与酶的变构调节) • Covalent modification of enzymes (酶的共价修饰) • Isoenzymes(同工酶) • Zymogens and activation of zymogens (酶原与酶原激活)

3. Outline and key points Enzyme structure* and the active site*** Quantitative analysis of E-catalyzed reaction rate *** How an enzyme facilitates the reaction? *** Enzyme activity can be regulated***

4. What are biocatalysts and enzymes ?

5. Biocatalysts and enzymes • Biocatalysts: catalyze every biological reactions --- enzymes --- ribozymes(核酶) and deoxyribozymes (脱氧核酶) • Enzymes typically are very large proteins, having highly efficient catalytic function • Ribozyme and deoxyribozyme: RNA and DNA with catalytic function

6. Section I Molecular structure and function of enzymes • Compositions of enzymes • Cofactors and coenzymes—辅助因子和辅酶*** • Active sites of enzymes---酶的活性中心*** • Isozymes---同工酶***

7. Some basic terms • Monomeric enzyme (单体酶): one polypeptide with tertiary structure • Oligomeric enzyme (寡聚酶): several subunits with quaternary structure • Multienzyme system (多酶体系): several enzymes cooperatively catalyze a reaction • Multifunctional enzyme (多功能酶): one enzyme contains a few catalytic domains

8. Molecular compositions of enzymes • Simple enzyme单纯酶: composed of protein only • e.g. Ribonuclease (核糖核酸酶), pepsin(胃蛋白酶) • Conjugated enzyme: 结合酶 • Protein + Non-protein components • cofactor (辅助因子)

9. Conjugated enzyme • Apoenzymes: determine enzyme specificity • Cofactors: required for an enzyme to be active Inactive Active Apoprotein

10. Cofactors • As a constituent of enzyme 酶的组成成分 • Function: activation of an enzyme • Classified as: -- coenzymes (辅酶) -- prosthetic groups (辅基)

11. Cofactors • Coenzymes --loosely bind to apoproteins --act as transient carriers of specific functional groups • Prosthetic groups -- tightly or even covalently bound to the apoproteins -- essential for a complete, catalytically active enzyme What are cofactors? metal ionsorsmall organic molecules

12. Metal ions • 2/3 enzymes contain Mg2+, Na+, Fe2+ , Zn2+… • Functions: • -- as catalytic groups • -- transfer of electrons • -- joining the substrate and enzyme • -- stabilization of enzyme conformation Substrate 底物: the molecule that is bound to the enzyme and acted upon by the enzyme

13. Metalloenzyme(金属酶): ions bind tightly to E • Metal activated enzyme (金属激活酶)：ions loosely bind to apoenzyme, easy to be removed

14. Small organic molecules • Functions: transport of electrons, H+ and chemical groups (-COOH, -CH3, -NH2…) • Common components: water-soluble vitamins and nucleotides

15. Nucleotides (核苷酸) Bases: A (腺嘌呤) G (鸟嘌呤) C (胞嘧啶) U (尿嘧啶) T (胸腺嘧啶)

16. Nucleotides (核苷酸)

17. Vitamins • Essential nutrients • lipid- soluble vitamins: A, D, E , K • water- soluble vitamins are main components of cofactors • B1, B2, B6, B12, C, Vitpp, biotin(生物素), pantothenic acid(泛酸), folic acid (叶酸) Chapter 5

18. Vit B1 and TPP TPP (Thiamine pyrophosphate 硫胺素焦磷酸酯) TPP: a coemzyme of some dehydrogenases (脱氢酶)

19. FAD：vitB2-nucleotide derivatives FlavinAdenine Dinucleotide B2= ribofalvin 核黄素

20. FMN and FAD FMN FMN= Flavin mononucleotide FAD

21. FMN and FAD--- transport of H2 FMNH2 (FADH2) FMN(FAD)

22. Vit B6 and phosphor-vitB6 Vit B6: 吡哆醛,吡哆胺,吡哆醇 Pyridoxal phosphate: coenzymes of aminotransferases (转氨酶)

23. Vit PP and NAD/NADP In NADP+, OHO-- P

24. NAD+/NADH+H+, NADP+/NADPH+H+-- Transport of H

25. Pantothenatic acid and Coenzyme A (CoA) Acetyl CoA: transfer acetyl group

26. Folic acid(叶酸) Active form-- FH4(THF): a carrier for one carbon unit 一碳单位的载体 Folic acid

27. Summary • Cofactors: • --required for an E (structure and activity) • --coenzymes, prosthetic groups • -- metal ions: Zn2+, Fe2+…… • --organic molecules: TPP, FMN/FMNH2, • FAD/FADH2, NAD/NADH……

28. Vitamins and their coenzyme forms

29. Vitamins and their coenzyme forms

30. How enzymes act as catalysts? • Enzyme(E)-catalyzed reactions are characterized by: • Binding to the substrates (S) • conversion of S to a product How an enzyme binds to its substrate(s) ?

31. Enzyme, substrate and catalytic site • Enzymes: typically huge proteins • Only a small part is actually involved in substrate binding and catalytic reaction

32. Active center(site) of enzyme*** 活性中心 • Active center, active site, catalytic site • Three-dimensional, local region of the enzyme, the region is composed of several essential groups of AAs, that has special spatial structure which specifically binds a substrate and catalyzes it to become product(产物) • Coenzymes can be involved in active site

33. Active center Essential groups Substrate Catalytic groups Binding groups An active site

34. Essential groups*** （必需基团） • Essential groups in the active sites --binding groups: bind S and coenzyme, and form an ES complex -- catalytic groups: catalyze the S to become product -- -OH, -COOH, -SH are usually essential groups in the active site • Essential groups outside of the active sites • maintenance of the spatial structure of active sites

35. Active site with the cAMP substrate cAMP is bound in active site with essential groups cAMP (red) is bound in deep cleft between large and small subunits

36. Active sites are often created at the interface(表面) between two domains of a protein or clefts on the protein surface

37. Steps in the process of enzyme catalysis • Binding of S in the active site, and forming an ES complex • Catalysis • Release of altered substrate = product

38. 乳酸脱氢酶 (lactate dehydrogenase, LDH) • LDH: Tetramer, contains two different subunits • M musel form • H heart form 乳酸 丙酮酸

39. Isozymes (isoenzymes, 同工酶) A group of enzymes: • catalyze the same reaction with similar structure of active site • have different protein structure, physicochemical and immunological properties • Encoded by different genes or different transcripts from the same gene • vary in different tissues

40. Clinical importance of isozymes • 可作为遗传标志，用于遗传分析 • 酶谱的改变有助于 • 对疾病的诊断 心肌梗死时，心肌细胞损伤，血液中LDH1, 2 (主要由心肌亚型亚基组成）活性显著增加 心肌梗死和正常人血清LDH同工酶谱的变化

41. Summary • In addition to catalytic RNAs/DNAs, all known enzymes are proteins • Many enzymes require cofactors for their catalysis • Water-soluble vitamins are main components of coenzymes or prosthetic groups • Substrate binding occurs in a pocket on the enzyme called the active site • E-catalyzed reactions are characterized by the formation of an ES complex

42. Section Ⅱ Mechanisms of Enzyme Action How an enzyme works? It increases the reaction rate by lowing activation energy

43. Enzymes, like other catalysts (催化剂): • increase the rate of reactions without themselves being altered in the process • do not determine the direction of a reaction • cannot alter the position of the equilibrium point • 不改变反应的平衡常数

44. Characteristics of enzyme-catalyzed reaction* Enzymes, unlike other catalysts: • High efficiency (高效) • High specificity (特异性强) • Enzyme activity can be regulated(活性可调节) • Enzyme activity is lost once the enzyme is denatured

45. High efficiency High efficiency：an enzyme molecule can process millions of molecules every second 转换数(turnover number，Kcat): 表示酶的催化效率，在酶被底物饱和时,每个酶分子每秒钟将底物转化为产物的分子数 Catalase(过氧化氢酶) kcat： 40,000,000 /sec One molecule of catalase can degrade (降解) 40,000,000 mol of H2O2 in one second

47. High specificity • The selectivity(选择性) of an enzyme for the substrate molecule which is catalyzed • Specificity depends on mutual(双方) structural recognition between enzyme (E) and substrate(S)

48. Specificity Absolutely specific绝对特异性 The E only reacts with a single substrate

49. Specificity Relatively Specific 相对特异性 An E works with similar molecules with the same functional group, or catalyzes a specific bond

50. Specificity Stereochemically specific 立体结构特异性 An E works with the proper stereo-isomer of a substrate