Biochemistry

1. Biochemistry Dept. of Biochemistry and Molecular Biology Professor Wu Yaosheng 2009-10

2. 還沒有來得及準備好接受這一地的金黃， 秋天就這樣悄無聲息的來到了我們的身邊。

3. Chapter 9 Regulation of Metabolism

4. Main Contents • Metabolic Regulation at Cell Level • Metabolic Regulation at Hormone Level • Regulation of Metabolism at Integral Level 4

5. Key Points and difficulties ◆ Some important metabolism molecules ◆ Key enzymes and their distribution ◆ Mutual relationship of carbohydrate, TG, Pr ◆ Regulation levels and fashion of substance metabolism 5

6. Digestion Absorption H2O Lipid Middle metabolism Sugar Salt Protein Vitamin Waste excretion Introduction Characteristics of Substance Metabolism 1.Mutual interknit among various metabolism pathways 各种物质代谢之间互有联系，相互依存。 6

7. 2. Metabolism processes regulated constant finely Inside and outside of circumstances To influence organism metabolism Subtle regulation mechanisms to regulate metabolism intensity, direction, velocity To fit in with the change of circumstances 7

8. 3. Various tissues and organs have themselves metabolism characters Different structures Different metabolism pathways Different organs Different enzymes and contents 8

9. Sugar digested and absorbed Various tissues Blood sugar glycogen degradation gluconeogensis 4. Each common metabolism pool • For example： 9

10. ADP+Pi To release energy Directly supply energy ATP 5. ATP is the common form for energy store and utilization Nutriment decomposition 10

11. 6. NADPH can supply the reduction equation for anabolism • For example： Pentose phosphate pathway NADPH + H+ Fatty acids, cholesterol Acetyl CoA 11

12. Questions 1. How to relate carbohydrate metabolism with lipid or protein metabolism by some important interim molecules? What are metabolic interrelationships? 2. What are the important significances of ATP during substance metabolism? 12

13. Section OneMetabolic Regulation at Cell Level

14. 1.1 Distribution of Enzymes in Cells • 代谢途径有关酶类常常组成多酶体系，分布于细胞的某一区域 。 14

15. Distribution of enzymes in main metabolic pathways 15

16. Distribution of enzymes in main metabolic pathways Compartmentalization of enzymes in cells Significances ◆To avoid interference among enzymes in different metabolic pathways ◆To be benefit to harmonious operation of enzymes 16

17. 1.2 Multienzyme system, Multifunctional Enzymes, and Isoenzymes 1.2.1 Multienzyme System and Multifunctional Enzymes Multienzyme system is an enzyme complex assembled by several different functional enzymes. For example, pyruvate dehydrogenase complex Multifunctional enzyme is an enzyme with different enzymatic functions in a single polypeptide. For example, fatty acid synthase system 17

18. The fatty acid synthase complex has 7 active sites: Acetyl CoA-ACP transacetylase (AT) b-ketoacyl-ACP synthase (KS) Malonyl CoA-ACP transferase (MT) b-ketoacyl-ACP reductase (KR) b-hydroxyacyl-ACP dehydratase (HD) Enoyl-ACP reductase (ER) Acyl carrier protein(ACP) 18

19. H H H H H H H H H M M M LDH1 (H4) LDH2 (H3M) LDH3 (H2M2) LDH4 (HM3) LDH5 (M4) H M M M M M M M 1.2.2 Isoenzymes Enzymes catalyzing the same reaction with different components and different physicochemical properties are named as isoenzymes. For example,LDH lactate dehydrogenase, LDH isoenyzmes 19

20. Example Two B M B M M B CK1(BB) CK2(MB) CK3(MM) brain cardiac muscle skeleton muscle 肌酸激酶 (creatine kinase, CK) 同工酶 20

21. 1.3 Basic Manners of Metabolic Regulation at Cell Level 1.3.1 Rate-Limiting Enzyme and Rated-Limiting Step Definition for rate-limiting enzyme: An enzyme with relatively low activity catalyzing the relatively low reaction speed for control the rate of the whole pathway is named rate-limiting enzyme. E1 E2 E5 E6 E3 E4 A B C D E F G 21

22. Rate-limiting enzymes of some metabolism pathways 22

23. (—) Glycogenphosphorylase Glucogenolysis : Gn G1P G6P G UDPG Glycogen synthase (+) 1.3.2 Feedback Regulation The end-products in metabolism pathways often affect the activities of the initial enzymes. Feedback regulation is one of the finest acting manners of regulatory enzymes. Negative feedback: most key enzymes Positive feedback: F-1,6-BP to 6-FPK-1 23

24. 1.3.3 Substrate Cycle Substrate cycle is the reversible interconversion between two substrates catalyzed by distinct enzymes for unilateral reactions. ATP ADP FPK-1 (+) (+) F-1,6-2P F-6-P AMP F-2,6-2P (–) (–) Pi Fructose biposphatase-1 24

25. 1.3.4 Cascade Reactions In a chain reaction, when an enzyme is activated, other enzymes are activated in turn to bring primal signal amplifying. 25

26. hormones（glucagon 、epinephrine）+ receptor Adenyly cyclase （inactive） Adenyly cyclase （active） ATP cAMP PKA (inactive) PKA (active) Phosphorylase b kinase Phosphorylase b kinase-P Phosphorylase b Phosphorylase a-P active inactive 26

27. 激素（胰高血糖素、肾上腺素等）+ 受体 腺苷环化酶 （无活性） 腺苷环化酶（有活性） ATP cAMP Pi PKA (无活性) PKA (有活性) 磷蛋白磷酸酶-1 磷酸化酶b激酶-P – 磷蛋白磷酸酶-1 磷蛋白磷酸酶-1 Pi Pi – – 磷蛋白磷酸酶抑制剂-P PKA（有活性） 磷酸化酶b激酶 糖原合酶 糖原合酶-P 磷酸化酶b 磷酸化酶a-P 磷蛋白磷酸酶抑制剂 27

28. 1.4 Regulation of Enzymatic Activity in Cells 1.4.1 Allosteric Regulation ( rapid regulation） when some metabolites combine reversibly to an regulating site of an enzyme and change the conformation of the enzyme, resulting in the change of enzyme activity. ◆allosteric enzyme ◆allosteric site Allosteric activator ◆allosteric effectors Allosteric inhibitor 28

29. Some allosteric enzymes and their effectors in metabolism pathways 29

30. General Properties of Allosteric Enzymes • Key points: • An allosteric enzyme is regulated by its effectors (activator or inhibitor). • Allosteric effectors bind noncovalently to the enzyme. • Allosteric enzymes are often multi-subunit proteins. • A plot of V0 against [S] for an allosteric enzyme gives a sigmoidal-shaped curve. • The binding of allosteric enzyme with an effector will induce a conformational change • Does not consume energy 30

31. FDP FDP FDP AMP AMP FDP FDP FDP FDP AMP AMP FDP Allosteric effect of fructose-1,6-biphosphatase AMP (allosteric inhibitor) Glyceraldehydes-3-phosphate FA –carrier protein (allosteric activator) T stateR state (high activity)(low activity) 31

32. 1.4.2 Covalent Modification (rapid regulation） It means the reversible covalent attachment of a chemical group. • Types of Covalent Modification: phosphorylation / dephosphorylation • adenylylation/deadenylylation • methylation/demethylation • acetylation/deacetylation • －SH / －S－S , etc 32

33. O- Protein-O-P=O O- Covalent Modification Protein-OH Pi ATP Protein phosphatase Protein kinase ADP H2O The reversible phosphorylation and dephosphorylation of an enzyme 33

34. Regulation of covalent modification in enzyme activities Enzyme Reactive type Effect PFK-1 Phosphorylation/dephosphorylationInactivity/activity Pyr DHasePhosphorylation/dephosphorylationInactivity/activity Pyr decarboxylasePhosphorylation/dephosphorylationInactivity/activity Glycogen phosphorylasePhosphorylation/dephosphorylation Activity/inactivity Phosphorylase b kinasePhosphorylation/dephosphorylationActivity/inactivity Protein phosphatasePhosphorylation/dephosphorylationInactivity/activity Glycogen synthasePhosphorylation/dephosphorylation Inactivity/activity Triacylglycerol lipasePhosphorylation/dephosphorylationActivity/inactivity HMG CoA reductasePhosphorylation/dephosphorylationInactivity/activity Acetyl CoA carboxylasePhosphorylation/dephosphorylationInactivity/activity 34

35. Key points: • The activity state of an enzyme modulated can interconvert reversely • Change of a covalent bond catalyzed by E, and can be modulated by hormones • The modification is a rapid, reversible and effective and amplified by cascade reaction • The most common is the phosphorylation or dephosphorylation. Enzymes----protein kinases or phosphatases 35

36. P P P P P P Covalent modification of phosphorylase Phosphorylase b kinase 2ATP 2ADP phosphatase 2Pi Phosphorylase b (dimer) Inactivity Phosphorylase a (dimer) High activity Phosphorylase a (tetramer) Activity 36

37. 1.5 Regulation of Enzyme Level in Cells (Genetic Control) The amount of enzyme present is a balance between the rates of its synthesis and degradation. The level of induction or repression of the gene encoding the enzyme, and the rate of degradation of its mRNA, will alter the rate of synthesis of the enzyme protein. Once the enzyme protein has been synthesized, the rate of its breakdown (half-life ) can also be altered as a means of regulating enzyme activity. 37

38. 1.5.1 Induction and repression of E Pr Synthesis Induction: the activation of enzyme synthesis. Repression: the shutdown of enzyme synthesis. Genetic control of enzyme leverl means to controlling the transcription of mRNA needed for an enzyme’s synthesis. In prokaryotic cells, it also involves regulatory proteins that induce or repress enzyme’s synthesis. Regulatory proteins bind to DNA, and then block or enhance the function of RNA polymerase. So, regulatory proteins may function as repressors or activators. 38

39. Repressor Repressors are regulatory proteins that block transcription of mRNA, by binding to the operator that lies downstream of promoter. This binding will prevent RNA polymerase from passing the operator and transcribing the coding sequence for the enzyme.------Negative control. Regulatory proteins are allosteric proteins. Some special molecules can bind to regulatory proteins and alter their conformation, and then affect their ability to bind to DNA. 39

40. For example: lac operon When no lactose: Operator gene Promotor Structural gene I A Y Z repressor gene RNA polymerase mRNA mRNA NH2 repressor protein 40

41. When lactose presents: Structural gene I O P A Y Z repressor gene RNA polymerase mRNA mRNA Z NH2 Y NH2 repressor protein A NH2 lactose 41

42. Inducers Inducers promote the transcription of mRNA. Activator is an allosteric protein which is unable to bind to promoter to transcribe relative genes directly in eukaryotes. When no inducer: Structural gene activator-binding site O P mRNA RNA polymerase Activator 42

43. When inducer: P O Structural gene activator-binding site mRNA RNA polymerase inducer activator 43

44. Bacteria also Use Translational Control of Enzyme Synthesis The bacteria produces antisense RNA that is complementary to the mRNA coding for the enzyme. When the antisense RNA binds to the mRNA by complementary base paring, the mRNA cannot be translated into protein. 44

45. 1.5.2 Degradation of Enzyme Proteins Cellular enzyme proteins are in a dynamic state with change of enzyme synthesis and degradation so that ultimately determine enzyme level at any point in time. In many instances, transcriptional regulation determines the concentrations of specific enzyme, with enzyme proteins degradation playing a minor role. In other instances, protein synthesis is constitutive, and the amounts of key enzymes and regulatory proteins are controlled via selective protein degradation. In addition, it also involves the abnormal enzyme proteins ( biosynthetic errors or post-synthetic damage). 45

46. There are two pathways to degrade enzyme protein in cells: 1. Lysosomal pathway ATP independent 2. Proteasome pathway ATP, Ubiquitin dependent 46

47. Questions 1. Which one of the following metabolism pathways is not present in cytoplasm? A. Glycolysis B. Phosphate pentose pathway C．Glycogenesis and glycogenolysis D．Fatty acid β-oxidation E．Fatty acid synthesis 47

48. Questions 2. All gluconeogenesis, ketone body biosynthesis and urea synthesis exist in A. Heart B．Kidney C．Brain D．Liver E．Muscle 48

49. Can you fill in these blanks? Substrate cycle is the reversible interconversion between two substrates catalyzed by distinct enzymes for unilateral reactions. ATP ADP ATP FPK-1 (+) (+) (+) (+) F-1,6-2P F-6-P AMP F-2,6-2P (–) (–) (–) (–) Pi Fructose biposphatase-1 Fructose biposphatase-1 49

50. Questions 1. Why some persons who are easely drunk can turn to endure alcohol after they have experience to drink wine? 2. Why some persons who need hypnotics (安眠药）would become more and more dependent to drugs? 50