生物化學(xué)：Chapter 15 Principles of Metabolic regulation

《生物化學(xué)：Chapter 15 Principles of Metabolic regulation》由會員分享，可在線閱讀，更多相關(guān)《生物化學(xué)：Chapter 15 Principles of Metabolic regulation（36頁珍藏版）》請在裝配圖網(wǎng)上搜索。

1、Chapter 15 Principles of Metabolic regulation as found in glucose metabolismBy Prof. Chang Zengyi (昌增益教授）昌增益教授）Biochemistry Lecture for Nov. 22, 2012The animal body was believed to maintain a constant internal environment (Milieu intrieur ) The constancy of the internal environment is the condition

2、for a free and independent life. The living body is relatively independent of its surrounding environment. This independence derives from the fact that the tissues are in fact withdrawn from direct external influences and are protected by a veritable internal environment which is constituted by the

3、fluids circulating in the body. All the vital mechanisms, varied as they are, have only one object, that of preserving constant the conditions of life in the internal environment.Claude Bernard(1813-1878) Bernard discovered glycogen as the sugar-forming substance“ in liver, whichgenerates glucose wh

4、en a dog was only fed with meat.The constancy of the body was considered as a dynamic balance “Physiological activity is constantly disturbing the internal environment. What is actually maintained is a dynamic balance between the disturbing and restorative activities (John Scott Haldane, 1922)Haldan

5、e, J. S. (1917). Organism and environment as illustrated by the physiology of breathing. Yale University Press.Haldane, J. S. (1922). Respiration. Yale University Press.Haldane, J. S. (1929). Claude Bernards conception of the internal environment. Science, 64, 453454.Haldane (18601936)Early observat

6、ions on hormone action to regulate animal physiology “Strong emotional states stimulate the secretion of adrenaline from the medulla of the adrenal gland and the hormone acts on peripheral tissues in such a way as to prepare the animal for vigorous action, either fighting or fleeing in life-threaten

7、ing emergencies.” (Walter Cannon, 1914).Cannon, W. B. (1914). The emergency function of the adrenal medulla in pain and the major emotions. American Journal of Physiology, 33, 356372.Canon(1871-1945)Animal body proposed to be homeostatic (dynamic steady state) Body constancy is maintained by certain

8、 mechanisms: any tendency toward change automatically meets with factors that resist change (Walter Canon, 1926). Homeostatic categories: Material supplies for cellular needs. 1. Glucose, protein, fat. 2. Water. 3. Sodium chloride and other inorganic constituents except calcium. 4. Calcium. 5. Oxyge

9、n. 6. Internal secretions having general and continuous effects. Environmental factors affecting cellular activity. 1. Osmotic pressure. 2. Temperature. 3. Hydrogen-ion concentration.Canon(1871-1945)Canon inBeijing (1935)Cannon WB. Organization For Physiological Homeostasis. Physiol Rev. 1929; 9: 39

10、9-431. W. B. Cannon. Physiological regulation of normal states: some tentative postulates concerning biological homeostatics. IN: A. Pettit (ed.). A Charles Richet: ses amis, ses collgues, ses lves, p. 91. Paris: ditions Mdicales, 1926. The feedback concept of electronics was applied in understandin

11、g regulation in living organisms First in hormone production (endocrinology, 1940s) and then in the biosynthesis of amino acids and nucleotides (1950s). Found to occur via allosteric regulation of an enzyme at the molecular level for biosynthesis.Wiener, N. (1948) Cybernetics, or the Control and Com

12、munication in the Animal and the Machine. New York: Wiley.Hoskins, R. G. (1949). The thyroid-pituitary apparatus as a servo (feed-back) mechanism. Journal of Clinical Endocrinology 9:1429-1451.Yates, R. A., and Pardee, A. B. (1956) Control of pyrimidine biosynthesis in Escherichia coli by a feedback

13、 mechanism. J. Biol. Chem. 221:757-770.Umbarger, H. E. (1956) Evidence for a negative-feedback mechanism in the biosynthesis of isoleucine. Science 123:848.Norbert Wiener(1894-1964) Molecular Homeostasis for metabolites achieved via dynamic regulation of catabolic and anabolic reaction networks All

14、the metabolic pathways are inextrically intertwined, forming a multidimensional network of reactions. Negative feedback regulation loops maintain the constancy. Understanding how the whole network is dynamically regulated is still a challenging issue.Both amount and catalytic activity of enzyme be r

15、egulated for controlling metabolismTime scale of regulation: milliseconds to seconds to hours.Many principles of metabolism regulation were learned by studying sugar metabolism Constancy of glucose level in blood (homeostasis). Organ cooperation in glucose uses and biosynthesis. Hormone (insulin, gl

16、ucagon, epinephrine) regulation of glucose metabolism. Allosteric regulation of glycogen phosphorylase activity by AMP. Reversible phosphorylation as a way to regulate glycogen phosphorylase activity.Yeast adjustment of glucose metabolism observed by Pasteur (1857) The Pasteur Effect: In the presenc

17、e of O2, yeast cells grew much faster, but far less glucose consumed! No significant changes in the concentrations of ATP or most of the intermediates. Hinted that cell metabolism is adjusted in response to conditions both inside and outside the cell.The flux through the glycolytic pathway is believ

18、ed to be regulated at three steps A step with a large negative change in free energy is assumed to be regulated (thus highly irreversible with a build-up of reactants). Three enzymes found to be regulated: hexokinase (step 1), phosphofructokinase-1 (step 3), and pyruvate kinase (step 10). The change

19、 in free energy for each step of glycolysis estimated from the concentration of metabolites in an erythrocyte. G = G + RT ln Q Glycogen phosphorylase found to be allosterically regulated (Cori & Cori, 1930s) Glycogen phosphorylase catalyzes the phosphorolysis of glycogen, using inorganic phosphate (

20、not ATP!), producing Glc 1-P. AMP (NOT cAMP) activates and ATP and Glc 6-P inactivate the muscle enzyme.Rabit muscle glycogen phosphorylase-AMP complex(a homodimer) Blue: glycogen binding site, Red: catalytic site Yellow: AMP allosteric siteOrange: phosporylated Ser14+ Pior + Glc 6-PNobel prize 1947

21、Cori ester Glycogen phosphorylase found to be reversibly phosphorylated (1950s) The active form is phosphorylated and the inactive form dephosphorylated, and such phosphorylation is regulated by hormones.Krebs, E. G., and Fischer, E. H. (1956) The phosphorylase b to a converting enzyme of rabbit ske

22、letal muscle. Biochim. Biophys. Acta 20, 150-157Edwin Krebs(1918-2009) Edmond Fischer(1920-, born in Shanghai ) Nobel prize 1992cAMP found to mediate liver glycogen phosphorylase activation by hormones When epinephrine or glucagon was added to intact dog liver cells, the phosphorylase activity found

23、 increased (by phosphorylation). cAMP was found to mediate this activation process.Sutherland E W, Rall T W. (1958) Fractionation and characterization of a cyclic adenine ribonucleotide formed by tissue particles. J Biol Chem. 232:1077-91.Nobel prize 1971Liver glycogen phosphorylase regulated by all

24、osteric effectors & reversible phosphorylation (cascades effecting a large amplification of the initial signal)PKA:cAMP dependent protein kinaseIntrinsic control Extrinsic control(Mobilize glucose in liver)PKA and PPI-1also regulate glycogensynthase, but reciprocally!Liver glycogen phosphorylase act

25、s as a glucose sensor Glucose binds to phosphorylase a, facilitating the action of the phosphatase and conversion to phosphorylase b (liver glycogenolysis stoped when blood glucose level is high). Glycogen phosphorylase a thus acts as the glucose sensor of the liver, slowing the breakdown of glycoge

26、n whenever the level of blood glucose is high. Glucose, but not AMP is an allosteric regulator!Only in liver: Glucose-6-phosphate + H2O glucose + PiGlycogen synthase was discovered and found to be also regulated by reversible phosphorylation (the phosphorylated form being inactive)!(Glycogen phospho

27、rylase does not catalyze glycogen synthesis!)Glycogen synthase uses UDP-glucose(not Glc-1-P!)Uridine diphosphate (UDP) found to be the carrier of active sugars (1950s) Originally identified as the thermostable coenzyme of Gal-1-P and Glc-1-P isomerization (in yeast). Then UDP-Glc found participating

28、 in trehalose, sucrose and glycogen (in liver and muscle) synthesis.Cardini, C. E., Paladini, A. C., Caputto, R., and Leloir, L. F. (1950) Uridine diphosphate glucose: the coenzyme of the galactose-glucose phosphate isomerization. Nature 165, 191193Leloir, L. F., and Cardini, C. E. (1957) Biosynthes

29、is of glycogen from uridine diphosphate glucose. J. Am. Chem. Soc. 79, 6340UDPNobel prize 1970Glycogenin, found as a self-glucosylating protein that primes glycogen synthesis (1984), provides a Tyr residue for attaching the first glucose residue in a glycogen molecule.UDP-glucose. Mn2+ Tyr194The gly

30、cogen synthase is phosphorylated at multiple sites by the action of multiple kinases (at least 11) Casein kinase II adds the first phosphoryl group (“priming”) before glycogen synthase kinase 3 can another 3.The human genome contains about 500 protein kinase genes (modifying about30% of the human pr

31、oteins)! The phosphorylated form is inactive and dephosphorylated form active. Glc-6-P and Glc activates glycogen synthase b.Glycogen synthase also regulated by reversible phosphorylation & allosteric effectorsA Glc-6-P sensorBinding of insulin to receptors (1) on cells then activates a cascade of p

32、rotein kinases (2) that cause the cells to take up glucose (3) and convert it into storage molecules as glycogen and fatty acids (4,5 6).(PKA for glucagon)PhosphorylatedCarbohydrate metabolism in liver asregulated by insulin and glucagon. Insulin promotes synthesis of glycogen;Epinephrine (in muscle

33、) and glucagon (in liver) promotes degradation of glycogen.Insulin activates but epinephrine inactivates phosphoprotein Phosphatase 1 (PP1), which in turn dephosphorylates glycogen phosphorylase kinase, glycogen phosphorylase, and glycogen synthaseGM - glycogentargeting protein(subunit of PP1)Well-f

34、ed state(hyperglycemia)Fasting state(hypoglycemia)Epinephrine stimulates glycogenolysis and glycolysis in muscle, suppresses glycolysis in liver.Preparing the animals for“fight-or-flight”Multiple enzymes are regulated in the glycolytic pathwayThe hexokinase isozymes in muscle and liver work for diff

35、erent purposes Muscle isozymes has low Km (0.1 mM) and allosterically inhibited by Glc 6-P. Liver isozyme has high Km (10 mM) and not inhibited by Glc-6-P but by a regulatory protein. Muscle isozyme(for ATP production)Liver isozyme(for blood glucosehomeostasis)Blood glucose level (5 mM)Hexokinase IV

36、 in liver prepares the high level ofblood glucose for glycogen and fatty acid synthesis.Activity of phosphofructokinase-1 regulated by negative & positive allosteric effectorsPFK-1 is inhibited by ATP (?) and citrate (?), but activated by fructose 2, 6-bisphosphate, AMP and ADP.Commits glucoseto gly

37、colysis(no longer availablefor PPP & glycogenesisActive siteAllosteric siteIt is doubtful whether ATP and citrate actaully play such regulatoryroles in living cells!Fructose 2, 6-bisphosphate found as a potent allosteric stimulator of liver phosphofructokinase-1 (1980) Study of mechanism of action o

38、f glucagon on liver gluconeogenesis led to the discovery. Concentration of a low-molecular-weight stimulator of phosphofructokinase greatly increased in hepatocytes in presence of glucose and decreased in presence of glucagon.Van Schaftingen, E., Hue, L. and Hers, H.-G. (1980) Control of the fructos

39、e-6-phosphate/fructose 1,6-bisphosphate cycle in isolated hepatocytes by glucose and glucagon. Role of a low-molecular-weight stimulator of phosphofructokinase. Biochem. J. 192, 887895Van Schaftingen, E., Hue, L. and Hers, H.-G. (1980) Fructose 2,6-bisphosphate, the probable structure of the glucose

40、- and glucagon-sensitive stimulator of phosphofructokinase. Biochem. J. 192, 897901Henri-Gery Hers(1923-2008- Belgium) Fructose 2, 6-bisphosphate found to inhibit fructose 1, 6-bisphosphatase and thus gluconeogenesisHenri-Gery Hers(1923-2008- Belgium) Level of F-2,6-BP set by the relative activities

41、 of phosphofructokinase-2 (PFK-2) and fructose 2, 6-bisphosphatase (FBPase-2) of a single bifunctional protein. It exhibits FBPase-2 activity when phosphorylated and PFK-2 activity when dephosphorylated (at a single Ser residue).Rapid hormonal regulation of glycolysis and gluconeogenesis is mediated

42、 by fructose 2, 6-bisphosphate Insulin promotes dephosphorylation, thus the increase of F2,6-BP. Glucagon promotes the phosphorylation, thus decrease of F2,6-BP;Observation in rat liver extract:Hexokinase and PFK-1 both contributeto setting the flux through the glycolytic pathway (hexokinasemore tha

43、n PFK-1!), and that phosphohexose isomerasedoes not The conventional simple solution of a single rate-determining seems to be wrong!Changes of enzymatic activity is now considered to serve two distinct but complementary roles Metabolic regulation: To maintain homeostasis at the molecular level, e.g.

44、, to keep concentration of a metabolite at a steady state level over time, even as the flow of metabolites through the pathway changes. Metabolic control: leading to a change in output of a metabolic pathway over time, in response to outside signal or change in circumstances.Metabolic control seems

45、to be contributed by many enzymes rather than confined to one rate-limiting enzyme per pathway. Whether an enzyme exercises strong control on a flux cannot be deduced solely from its own properties, nor is it directly related to its distance from equilibrium. An enzyme may show large changes in acti

46、vity (i.e.highly regulated) but if these changes have little effect on the flux of a metabolic pathway, then this enzyme is not involved in the control of the pathway! Regulation of metabolism is dynamic and occurs in a network of reactions and the study of which is undoubtedly greatly challenging.

47、Mathematics and systems methods (modeling) would be essential to understand the metabolic control in such multi-dimensional net works of metabolic pathways. Metabolic network of the Arabidopsis thaliana citric acid cycleEnzymes and metabolites are shown as red squares and the interactions between them as black lines.