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Biochemistry Dept. of Biochemistry and Molecular Biology Professor Wu Yaosheng 2009-10 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。 2 Chapter 9 Regulation of Metabolism Main Contents 1. Metabolic Regulation at Cell Level 2. Metabolic Regulation at Hormone Level 3. Regulation of Metabolism at Integral Level 4 4 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 5 Introduction Characteristics of Substance Metabolism 1.Mutual interknit among various metabolism pathways Digestion Absorption Lipid Sugar H2O Salt Protein Vitamin Middle metabolism Waste excretion 各种物质代谢之间互有联系，相互依存。 6 6 2. Metabolism processes regulated constant finely Inside and outside of circumstances To fit in with the change of circumstances To influence organism metabolism Subtle regulation mechanisms to regulate metabolism intensity, direction, velocity 7 7 3. Various tissues and organs have themselves metabolism characters Different structures Different organs Different metabolism pathways Different enzymes and contents 8 8 4. Each common metabolism pool For example： gluconeogensis Various tissues glycogen degradation Blood sugar Sugar digested and absorbed 9 9 5. ATP is the common form for energy store and utilization To release energy ADP+Pi Directly supply energy Nutriment decomposition ATP 10 10 6. NADPH can supply the reduction equation for anabolism For example： Pentose phosphate pathway NADPH + H+ Acetyl CoA Fatty acids, cholesterol 11 11 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 12 Section One Metabolic Regulation at Cell Level 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 14 Distribution of enzymes in main metabolic pathways Metabolic pathways Distribution Metabolic pathways Distribution Glycolysis Cytosol Oxidation phosphorylation Mitochondrion Citric acid cycle Mitochondrion Protein synthesis ER Pentose phosphate pathway Cytosol Urea synthesis Mitochondrion, cytosol Gluconeogenesis Cytosol DNA synthesis Nucleus Glycogenesis and glycogenolysis Cytosol mRNA synthesis Nucleus Fatty acid β-oxidation Mitochondrion tRNA synthesis Nucleoplasm Fatty acid synthesis Cytosol rRNA synthesis Nucleus Respiratory chain Mitochondrion Heme synthesis Cytosol, Mitochon. Phospholipid synthesis Endoplasmic reticulum Hydrolytic enzymes Lysosome Cholesterol synthesis ER, Cytosol Bilirubin synthesis ER, cytosol 15 15 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 16 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 17 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 18 1.2.2 Isoenzymes Enzymes catalyzing the same reaction with different components and different physicochemical properties are named as isoenzymes. For example, LDH H H H H H H H M M M H H H M M M M M M M LDH1 (H4) LDH2 (H3M) LDH3 (H2M2) LDH4 (HM3) LDH5 (M4) lactate dehydrogenase, LDH isoenyzmes 19 19 Example Two B B M B CK1(BB) CK2(MB) brain M M CK3(MM) cardiac muscle skeleton muscle 肌酸激酶 (creatine kinase, CK) 同工酶 20 20 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. A E1 B E2 C E3 D E4 E E5 F E6 G 21 21 Rate-limiting enzymes of some metabolism pathways Metabolism pathway Rate-limiting enzymes Glycolysis HK , PFK-1, PK P.P.P G6PD Gluconeogenesis Pyr carboxylase, PEP carboxykinse, FBPase, G6Pase Cictric acid cycle Citrate synthase, Isocitrate DHase, α-KG DHase Glycogenesis Glycogen synthase Glycogenolysis Glycogen phosphorylase Triacylglycerol hydrolysis Triacylglycerol lipase FA synthesis Acetyl CoA carboxylase Ketogenesis HMG CoA synthase Cholesterol synthesis HMG CoA reductase Urea synthesis Argininosuccinate synthase Heme synthesis ALA synthase 22 22 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 Glucogenolysis : Gn Glycogen synthase Glycogen phosphorylase UDPG (—) G1P G6P G (+) 23 23 1.3.3 Substrate Cycle Substrate cycle is the reversible interconversion between two substrates catalyzed by distinct enzymes for unilateral reactions. ATP (+) F-6-P AMP (–) Pi FPK-1 ADP (+) F-2,6-2P F-1,6-2P (–) Fructose biposphatase-1 24 24 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 25 hormones（glucagon 、epinephrine）+ receptor Adenyly cyclase （inactive） Adenyly cyclase （active） ATP cAMP PKA (inactive) Phosphorylase b kinase Phosphorylase b inactive PKA (active) Phosphorylase b kinase-P Phosphorylase a-P active 26 26 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 磷酸化酶b激酶 PKA PKA (无活性) (有活性) 磷蛋白磷酸酶-1 磷酸化酶b激酶-P 糖原合酶 Pi 糖原合酶-P 磷酸化酶b 磷蛋白磷酸酶-1 – Pi – 磷酸化酶a-P 磷蛋白磷酸酶-1 – 磷蛋白磷酸酶抑制剂-P PKA（有活性） 磷蛋白磷酸酶抑制剂 27 27 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 28 Some allosteric enzymes and their effectors in metabolism pathways Metabolism Allosteric enzymes Activator HK Glycolysis Inhibitor G-6-P 6-FPK-1 AMP, ADP, F-1,6-BP, F-2,6-BP Citrate, ATP Pyruvate kinase F-1,6-BP ATP, alanine Citrate synthase ADP ATP, citrate, NADH Isocitrate dehydrogenase ADP ATP, Ca2+ Pyruvate carboxylase Acetyl CoA ADP F-1,6-bisphosphatase Citrate AMP, F-2,6-BP Glycogenolysis Glycogen phophorylase b AMP, G-1-P, Pi ATP, G-6-P Glycogenesis Glycogen sythase G-6-P FA biosynthesis Acetyl CoA carboxylase Citrate, isocitrate Cholesterol biosynthesis HMG-CoA carboxylase AA metabolism L-glutamate dehydrogenase ADP, leucine, methionine ATP, GTP, NADH Purine synthesis PRPP amidotransferase PRPP AMP, ADP, GMP, GDP, Pyrimidine synthesis Aspartate transcarbomoylase CTP ALA synthase Heme Citric acid cycle Gluconeogenesis Heme synthesis Long-chain fatty acyl-CoA Cholesterol 29 29 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 30 Allosteric effect of fructose-1,6-biphosphatase FDP FDP FDP FDP FDP AMP (allosteric inhibitor) AMP FDP FDP Glyceraldehydes-3-phosphate FA –carrier protein (allosteric activator) AMP T state (high activity) AMP AMP FDP R state (low activity) 31 31 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 32 Covalent Modification Pi Protein phosphatase H2 O Protein-OH O- ATP Protein kinase Protein-O-P=O O- ADP The reversible phosphorylation and dephosphorylation of an enzyme 33 33 Regulation of covalent modification in enzyme activities Enzyme Reactive type Effect PFK-1 Phosphorylation/dephosphorylation Inactivity/activity Pyr DHase Phosphorylation/dephosphorylation Inactivity/activity Pyr decarboxylase Phosphorylation/dephosphorylation Inactivity/activity Glycogen phosphorylase Phosphorylation/dephosphorylation Activity/inactivity Phosphorylase b kinase Phosphorylation/dephosphorylation Activity/inactivity Protein phosphatase Phosphorylation/dephosphorylation Inactivity/activity Glycogen synthase Phosphorylation/dephosphorylation Inactivity/activity Triacylglycerol lipase HMG CoA reductase Phosphorylation/dephosphorylation Phosphorylation/dephosphorylation Acetyl CoA carboxylase Phosphorylation/dephosphorylation Activity/inactivity Inactivity/activity Inactivity/activity 34 34 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 35 Covalent modification of phosphorylase 2ATP Phosphorylase b kinase 2Pi phosphatase Phosphorylase b (dimer) Inactivity 2ADP P P Phosphorylase a (dimer) High activity P P P P Phosphorylase a (tetramer) Activity 36 36 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 37 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 38 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 39 For example: lac operon When no lactose: Promotor Operator gene Structural gene I Z repressor gene A RNA polymerase mRNA mRNA repressor protein Y NH2 40 40 When lactose presents: I repressor gene P O Structural gene A Y Z RNA polymerase mRNA mRNA NH2 NH2 NH2 repressor protein Z Y A lactose 41 41 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: activator-binding site P Structural gene O mRNA Activator RNA polymerase 42 42 When inducer: activator-binding site P Structural gene O mRNA RNA polymerase activator inducer 43 43 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 44 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 45 There are two pathways to degrade enzyme protein in cells: 1. Lysosomal pathway ATP independent 2. Proteasome pathway ATP, Ubiquitin dependent 46 46 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 47 Questions 2. All gluconeogenesis, ketone body biosynthesis and urea synthesis exist in A. Heart B．Kidney C．Brain D．Liver E．Muscle 48 48 Can you fill in these blanks? Substrate cycle is the reversible interconversion between two substrates catalyzed by distinct enzymes for unilateral reactions. ATP (+) F-6-P AMP (–) Pi FPK-1 ADP (+) F-2,6-2P F-1,6-2P (–) Fructose biposphatase-1 49 49 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 50 Section Two Metabolic Regulation at Hormone Level Hormones are generally secreted by endocrine glands, travelled by blood stream to specific target cells. By these mechanisms, hormones regulate the metabolic processes in various organs and tissues; facilitate and control growth, differentiation, reproductive activities, learning and memory; and help organisms coping with changing conditions and stresses to around environment. 52 52 Hormonal regulation depends upon the transduction of the hormonal signal across the plasma membrane to specific intracellular sites, particularly the nucleus. Many steps in these signal across the signalling pathway involve phosphorylation of Ser, Thr, and Tyr residues on target proteins. According to receptor’s location in a cell, hormones are divided into two classes: Hormones act on cell membrane receptors Hormones act on intracellular receptors 53 53 Hormones act on cell membrane receptors 54 54 Hormones act on intracellular receptors 55 55 2.1 Regulation of Hormones to Receptors on Cell Membrane Hormones act on membrane receptors, as the first messenger, to activate various signal transduction pathways that mobilize various second messengers----cAMP, cGMP, Ca2+, IP3 , DG that activate or inhibit enzymes or cascade of enzymes in specific ways. The first messengers: Peptide or protein hormones: GH, Insulin, etc Amino acid derivatives: epinephrine, norepinephrine 56 56 H Adenylate cyclase cAMP Ｒ R β β γ α γ AA CC GDP GTP ATP 57 57 Hormone receptor Ｇ protein Enzyme The second messenger Protein kinase Enzyme or other protein Biological effects 58 58 2.2 Regulation of Hormones to Receptors in Cells Hormones to act on intracellular receptors: Steroid hormones: Glucocorticoids Mineralocorticoids Vit D Sex hormones Amino acid derivatives: T3, T4 59 59 60 60 Hormone Can you give some examples? receptor Ｇ protein Enzyme The second messenger Protein kinase Enzyme or other protein Biological effects 61 61 Section Three Regulation of Metabolism at Integral Level Living in a constantly changing environment, human must have the ability to adapting to the environment. Why and how? The metabolism of body has to be regulated through neurohumoral pathways to satisfy energy needs and to maintain homeostasis of the internal environment. 63 63 3.1 Metabolism Regulation in Starvation 3.1.1 Starvation in Short-term (1-3 days) Glycogen reserve Blood Glucose Insulin glucagon corticosteroid a series of metabolic changes 64 64 (1) Protein Metabolism Protein degradation ↑, Amino acid Protein Glucose degradation gluconeogenesis Amino acid Pyruvate deamination transamination Pyruvate transamination Alanine Muscle Glucose Alanine Liver Blood 65 65 (2) Carbohydrate Metabolism Gluconeogenesis Lactic acid 30% Glycerol 10% Amino acids 40% Liver : 80% Renocortical : 20% Tissue utilize glucose In brain , glucose is still the main fuel source. 66 66 (3) Triacylglycerol Metabolism Fat mobilization Fatty acid Ketone bodies Heart Skeletal muscle Renal cortex 67 67 3.1.2 Change of Metabolism in Long-term Starvation ( >7 days) 68 68 Starvation in Long-term (1) Protein Metabolism Muscle protein degradation Amino acid , but Glu deamination In urine Urea NH3 Acidism(酸中毒） ( by ketosis 酮症) 69 69 (2) Carbohydrate Metabolism In kidney : Gluconeogenesis ( almost equal to that in liver ) The main materials of gluconeogenesis in liver: Lactic acid Pyruvate 70 (3) Triacylglycerol Metabolism Fat mobilization Fatty acid Ketone bodies Skeletal muscle: FA as an energy source to ensure that adequate amounts of ketone bodies are available in brain. Brain: gradually adapts to using ketone bodies as fuel. This may reduce utilization of glucose and gluconeogenesis of amino acid, so decrease the breakdown of protein. 71 71 After starvation in Long-term, if the person is given a big meal with a lot of meat and wine in short time, what case would occur? 72 72 3.2 Metabolism Regulation in Stress Stress is a tense state of an organism in response to unusual stimulus. Effect: Stimulus injury Excitation of sympathetic nerves pain Adrenal medullary/cortical hormones frostbite Epinephrine, glucagons, growth hormone oxygen deficiency Insulin toxicosis Metabolism of carbohydrates infection lipids out-of-control rage proteoins Catabolism change Anabolism 73 73 (1) Change of Carbohydrate Metabolism Hyperglycemia catecholamine glucagon growth hormone corticosteroid Glycogenolysis Gluconeogenesis Stress hyperglycemia Stress glucosuria Insulin Blood glucose If exceeds renal threshold of glucose (8.96 mmol/L) Glucosuria 74 74 (2) Change of Triacylglycerol Metabolism Adrenaline Noradrenaline Glucagon Fat mobilization Fatty acid Ketone bodies Tissue utilize FA as energy 75 75 (3) Change of Protein Metabolism Protein hydrolysis Amino acid: as material for Gluconeogenesis Urea synthesis Equilibrium of negative nitrogen 76 76 Liver Glycogenolysis Glycerophosphate Ketogenesis Stress Sympathetic excitation Adrenal cortex/ medulla hormone FA LA glucose Gluconeogenesis Pyruvate Ureogenesis Alanine NH3 FA LA Alanine Urea Glucose Glycerophosphate Kidney Blood vessel Glucosuria TG hydrolysis Lipocyte Muscle glycogenolysis Muscle Protein degradation 77 77 Questions 1. Which one of substance change in blood is incorrect under stress ? A. Glucose increase B. Free fatty acid increase C．Amino acid increase D．Ketone body increase E．VLDL increase 78 78 Questions 2. When hungry, the false statement about substance metabolism alternation is A．Gluconeogenesis enhancement B. Triglyceride mobilization enhancement C．Ketone body synthesis enhancement D．Insulin secretion increase E. Glucagon secretion increase 79 79 Questions 1.How does Ala turn to be glucose in vivo? When does this case occur？ 2. How does carbohydrate metabolism and amino acid metabolism be modulated in liver cells to adapt with those in skeleton muscles and in cardiac muscle? 80 80 Questions 3. How to compare allosteric regulation with chemical modification? 4. Use several examples to explain some diseases involved with abnormal metabolism. 5. What changes of metabolism in body would occur in long-term starvation? 81 81