Class Note for BIOC 460 at UA 2
Class Note for BIOC 460 at UA 2
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Date Created: 02/06/15
Biochemistry 460 Dr Tischler GLYCOLYSIS AND GLUCONEOGENESISZ Related Reading Chapter 16 452470 in Stryer 6m edition OBJECTIVES Gluconeogenesis Pathway 1 Identify the key gluconeogenic precursors in liver and write out the four reactions that are unique to gluconeogenesis 2 Explain the signi cance of the glyceraldehyde 3 phosphate dehydrogenase and phosphoglycerate kinase reactions in gluconeogenesis Regulation of lecolvsis and Gluconeogenesis 1 Compare and contrast the regulation of glucokinase and hexokinase and explain the logic for these differences between the two kinases 2 In relation to regulation of phosphofructokinase l and fructose 16 bisphosphatase a compare their allosteric regulation b describe the pathway for metabolizing fructose26bisphosphate and explain the role of the bifunctional protein in this pathway c describe how insulin via fructose26bisphosphate ensures during the fed state that liver glycolysis is active and that gluconeogenesis is inactive 3 Discuss the regulation of pyruvate kinase via allosteric control and by covalent modification phosphorylaiion dephosphorylaiion PHYSIOLOGICAL PREMISE The regulation of carbohydrate pathways serves multiple purposes Ultimately most allosteric control is designed to meet the physiological needs of the cell rather than fuel requirements of the organism as a whole Logically this is so because if the cell fails to function normally it is unable to participate in fuel homeostasis Intracellular regulation has important implications both for the physiologic maintenance of blood glucose homeostasis and for balancing carbohydrate metabolism in individual cells In the fed state regulation helps store the excess glucose from the diet with hormonal signals being very signi cant In liver any glycogen used during a preceding period of food deprivation must be replenished In muscle any glycogen used during exercise must be replaced though this process does not depend necessarily on the fed state because residual lactate can be converted back to glycogen Once hepatic glycogen has been replaced the remaining excess glucose is converted to fatty acids in both liver and adipose tissue In a later lecture we will discuss the process of fatty acid biosynthesis lipogenesis but it is important to note that glucose must be processed through glycolysis and then pyruvate dehydrogenase to provide the acetyl CoA required for lipogenesis Hence the regulation of glycolysis and pyruvate dehydrogenase will in part determine whether lipogenesis can occur at least to the extent of providing precursors Glycolysisgluconeogenesis 2l In starvation mobilization of glycogen glycogenolysis and activation of glucose biosynthesis gluconeogenesis must occur Concurrently the opposing pathways of glycogenesis and glycolysis must be inhibited in the liver Hormonal signaling initiates these events but some allosteric control also takes place SYNTHESIS OF GLUCOSE GLUCONEOGENESIS Overview Gluconeogenesis is the synthesis of carbohydrate from noncarbohydrate precursors These precursors in the liver include primarily lactate and alanine Most of the remaining amino acids can also be used to synthesize glucose in starvation after being liberated from muscle protein Additionally glycerol derived from triacylglycerol stores that are mobilized in starvation is also a precursor to glucose see Fig l Gluconeogenesis uses reactions in both the mitochondria and in the cytoplasm Thus mitochondrial transport systems serve a role in this metabolic pathway Although gluconeogenesis occurs largely in the liver the kidney makes some contribution during starvation In kidney glutamine released from muscle is the primary precursor to glucose In starvation when liver glycogen is depleted about 18 to 24 h after the last meal gluconeogenesis becomes essential for maintaining blood glucose homeostasis Even though muscle cannot synthesize glucose it contains all of the enzymes of this pathway except for the last step and uses the pathway for converting lactate to glycogen instead of lactate to glucose Four Reactions Unique to Gluconeogenesis Figure 1 Unique Reaction 1 Pyruvate carboxylase catalyzes the reaction pyruvate ATP C02 9 oxaloacetate ADP Pi O OH O OH gt O O i H3 H2 OC OH Like all carboxylases this enzyme requires biotin as a prosthetic group to carry C02 and requires ATP to drive the reaction This is the rst of three sites of direct energy utilization for gluconeogenesis Remember that glucose contains siX carbons so that two molecules of ATP will be consumed at this step for each molecule of glucose synthesized from pyruvate This reaction is obligatory only for those precursors that must be converted to glucose via pyruvate We will discuss later the additional role of this enzyme in the fed state for lipogenesis Unique Reaction 2 Phosphoenolpyruvate carboxykinase PEPCK catalyzes the reaction oxaloacetate GTP 9 phosphoenolpyruvate GDP C02 0 OH i 0 OH 0 gt OP01239 H2 H 2 0C OH PEPCK is the ratelimiting and committed step of gluconeogenesis This reaction utilizes GTP as an energy source and as for pyruvate carboxylase two molecules are consumed for each molecule of glucose that is synthesized Amino acid precursors of glucose that are rst converted to a citric acid cycle intermediate eg glutamate is converted to xketoglutarate aspartate to oxaloacetate tyrosine to fumarate use PEPCK as the rst Glycolysisgluconeogenesis 22 reaction of gluconeogenesis During starvation the quantity of this enzyme is increased in liver cells to facilitate the synthesis of large amounts of glucose On refeeding the enzyme disappears because glucose synthesis is no longer required Unique Reaction 3 Fructose 16 bisphosphatase Fl6BPase catalyzes the reaction fructose16bisphosphate 9 fructose6phosphate P CHZOPOf39 CH20P03239 O HZOPOf39 O HZOH k H k H OH OH OH OH The reaction is irreversible and opposes phosphofructokinasel fructose6phosphate ATP 9 fructose16 bisP in glycolysis see previous lecture If these two enzymes were not regulated in an opposite manner as discussed later in this lecture ATP would be lost This would occur because ATP is hydrolyzed by phosphofructokinasel but is not produced by fructose16biphosphatase that releases phosphate Consequently energy would be lost Unique Reaction 4 Glucose G phosphatase G6Pase catalyzes the reaction glucose6phosphate H20 9 glucose P CHZOP03239 CHon H gt H OH OHOH OH OHOH The reaction is only found in the two tissues that can produce glucose liver and kidney Glucose6phosphate is transported into the endoplasmic reticulum not shown in the gure for removal of the phosphate by hydrolysis Glucose and P are then returned to the cytoplasm Glucose is exported to the blood and P remains in the cell Other Key Reactions in Glucaneagenesis see glycolysis for review The remaining reactions in gluconeogenesis are the reverse of glycolytic reactions Fig l Phosphoglycerate kinase ATP 3Phosphoglycerate 9 13Bisphosphoglycerate ADP This reaction is signi cant because it is an energy requiring step in gluconeogenesis whereas it is energy producing step in glycolysis Glyceraldehyde 3 phosphate dehydrogenase 13Binglycerate NADH 9 Glyceraldehyde3P NAD This reaction requires NADH without which gluconeogenesis cannot occur Lactate is unique among the gluconeogenic precursors in that its metabolism via lactate dehydrogenase generates the NADH required for gluconeogenesis Other precursors require that NADH be generated by processing in the mitochondria Glycolysisgluconeogenesis 23 Figure 1 Gluconeogenic pathways for conversion of lactate alanine aspartate and glycerol to glucose Bolded enzymes are unique to gluconeogenesis PC pyruvate carboxylase PEPCK phosphoenolpyruvate carboxykinase F16BPase fructosel6bisphosphatase G6Pase glucose6phosphatase Steps between PEP phosphoenolpyruvate and fructose16bisP Fl6BP are the reverse of glycolysis including the signi cant enzymes PGK phosphoglycerate kinase and G3PDH glyceraldehyde3phosphate dehydrogenase because of their requirements for ATP and NADH respectively The cofactors and other substrates for each reaction are described in the teXt Other enzymes used for speci c precursors are ALT alanine transaminase AST aspartate transaminase LDH lactate dehydrogenase Transaminases involve the transfer of the amino group of the amino acid to a keto group of an ocketoglutarate to form glutamate and the ocketoacid form of the amino acid pyruvate from alanine oxaloacetate OAA from aspartate Glycerol is converted to DHAP dihydroxyacetonephosphate that combines with G3P glyceraldehyde3phosphate to form Fl6BP G6Pase Glucose 6P Gucose Fructose 6P F1 6BPase FtGBP G3P DHAP Glycerol Lactate NAD NAD G3P DH NADH LDH ADP Pi Alanlne PGK NADH ALT ATP Pyruvate T PEP CYTOPLASM J PC PEPCK Pyruvate OAA gt PEP MITOCHONDRION 3T Aspartate Glycolysisgluconeogenesis 24 REGULATION OF GLUCOSE PHOSPHORYLATION Although hexokinase glucokinase represents the initial step of the glycolytic pathway their regulation is presented separately because glucose6phosphate may enter glycogenesis or gluconeogenesis as well as glycolysis Hence the regulation of these enzymes has implications for all carbohydrate pathways Comparative Kinetics of Hexokinase and Glucokinase Hexokinase which phosphorylates all hexose sugars though it most frequently acts on glucose is found in most tissues except liver and pancreas The enzyme is maximally active even at fasting glucose concentrations because ofits low Km for glucose This is important because cells such as brain or red blood cells need to process glucose from energy under all nutritional states The one exception to this occurs when the brain uses ketone bodies in prolonged starvation That hexokinase can phosphorylate glucose in muscle and adipose cells even at fasting blood glucose concentrations does not present a problem for blood glucose homeostasis because uptake of glucose by these cells depends on insulin which is diminished in starvation In contrast glucose uptake by brain does not depend on insulin so that hexokinase in this tissue rapidly phosphorylates glucose even in starvation In contrast to hexokinase glucokinase which is speci c for glucose is found solely in the liver and betacells of the pancreas Liver contains speci c kinases for other sugars see preceding Glycolysis lecture whereas the pancreas speci cally metabolizes glucose but not other hexoses Because it has a high Km glucokinase responds to only high blood glucose concentrations such as after a meal thus providing kinetic control At fasting concentrations of blood glucose 35 leI the rate of glucokinase is very low thus preventing glucose use by the liver and preventing glucose from promoting insulin secretion from the pancreatic Bcells Control of Glucose Phosphorylaiion Hexose eg glucose Glucose fruldotse Figure 2 Regulation of glucokinase and ya ac ose ATP I hexok1nase Glucokinase Hackr7359 no allosteric feedback control inhibition ADP I by GGP GlucoseGphosphate HexoseGphosphaie liver pancreas primarily GGP most tissues Hexokinase but not glucokinase undergoes feedback inhibition by its product glucose G phosphate Fig 2 This inhibition prevents the buildup of phosphorylated glycolytic intermediates that would trap phosphate needed for synthesis of ATP As will be discussed below excess glucose6phosphate also promotes the storage of glycogen for the same reason In the brain glucose6phosphate does not accumulate because the very high energy demands keeps glycolysis operating at a maximal rate Glucokinase is not regulated by glucose6 phosphate This occurs because it is imperative that when glucose is present in large amounts that the liver and pancreatic betacells continue to process glucose to prevent hyperglycemia In the liver the excess glucose is converted to fat In the pancreas the high glucose ensures the release of insulin Glycolysisgluconeogenesis 25 REGULATION OF GLYCOLYSIS AND GLUCONEOGENESIS Regulation 0fPh0sph0fmct0kinase 1 and Fructose16bisph0sphatase Figure 3 Fructose 16 bisphosphatase forms a futile cycle with phosphofructokinasel because if both reactions operate simultaneously there is a net destruction of ATP with the production of heat This is exactly what happens naturally with the shivering re ex The muscle stimulation related to shivering causes the loss of control of these enzymes so that heat is produced Regulation by AMP In muscle the critical regulator is AMP AMP is a signal of low energy in the muscle cell because its concentration increases when this occurs as in exercise In such situations the rate of glycolysis must increase to meet energy demands by metabolizing more glucose Accordingly AMP allosterically activates phosphofructokinasel When energy levels are high then the amount of AMP becomes very low and ATP inhibits phosphofructokinasel Since gluconeogenesis is an energyrequiring pathway it makes sense that AMP allosterically inactivates fructose16bisphosphatase to prevent the wasteful use of ATP Regulation by pH Changes of intracellular pH also affect glycolysis During active anaerobic carbohydrate metabolism in muscle acid is produced To prevent an excessive decline of the pH in the cell due to an elevated proton concentration high amounts of If inhibit phosphofructokinasel to slow the further production of acid Recall that the production of acid by metabolism favors the dissociation of oxygen from hemoglobin to boost aerobic metabolism Regulation by citrate In liver and muscle citrate is a feedback inhibitor of glycolysis by allosterically inactivating phosphofructokinasel Excess citrate is a signal that carbon is accumulating in the cell Since citrate is derived ultimately from glucose carbons it makes sense to shut off this supply when citrate is abundant Activation of fructosel6bisphosphatase by citrate can promote the conversion of these carbons back to glucose in liver Pi ATP Fructose6phosphate Citrate PFK1 ATP AMP F16BPase H fructose26bIsP gluconeogenesis glycolysis AMP Cltrate fructose26bisP ADP Fructose16bisphosphate Figure 3 Reciprocal regulation of glycolysis and gluconeogenesis at reactions catalyzed by phosphofructokinasel and fructosel6bisphosphatase Role ofFructose 2 6 bisphosphate in Carbohydrate Regulation in Liver Regulation In liver the most important allosteric regulator of these enzymes is fructose26bisphosphate While the role of most allosteric regulators is to bene t the cell fructose26bisphosphate is an exception Its intracellular concentration is related to the concentration of glucose in the blood When blood glucose levels Glycolysisgluconeogenesis 26 increase after a meal then fructose26bisphosphate is produced see below The fructose26bisphosphate then activates phosphofructokinasel to promote use of glucose in liver glycolysis to lower the blood concentration of glucose Simultaneously fructose26bisphosphate inhibits fructose16bisphosphatase gluconeogenesis to prevent liver from producing glucose With food deprivation the lack of fructose26bisphosphate in the liver cell reverses these events so that glycolysis is no longer activated and gluconeogenesis is no longer inhibited This indirect way of activating gluconeogenesis allows for the synthesis of glucose in liver when dietary glucose is no longer available Metabolism of fructose 26 bisphosphate There are two enzyme activities that are responsible for the metabolism of fructose26bisphosphate phosphofructokinaseZ and fructose26bisphosphatase Fig 4 Note do not confuse these with the reactions phosphofructokinasel and fructosel6bisphosphatase that are found in the glycolytic and gluconeogenic pathways respectively It is noteworthy that these two enzyme activities are contained in a single protein molecule Because the protein has two enzyme activities it is termed a bifunctional protein The advantage of a single protein containing two enzymatic activities is that regulation of the single protein controls both activities simultaneously Thus only one activity can function at any given time In the fed state the bifunctional protein remains unmodi ed no phosphorylation and in this circumstance the phosphofructokinaseZ is active whereas fructose26bisphosphatase has no activity Fig 4 top Consequently in the fed state the bifunctional protein catalyzes the formation of fructose26bisphosphate In contrast when the bifunctional protein is covalently modi ed by phosphate in a reaction catalyzed by protein kinase A see G protein lecture then fructose26bisphosphatase becomes active while phosphofructokinaseZ activity is suppressed Fig 4 bottom In this instance the concentration of fructose26bisphosphate in the cell markedly diminishes fructose6phosphate ATP Bifunctional protein dephosphorylated PFK Z active FBPase 2 inactive ADP Figure 4 Reactions in the metabolism of fructose26bisphosphate The formation frucmse39z 39bisphosphate of fructose26bisphosphate is catalyzed by phosphofructokinaseZ PFK2 and the conversion back to fructose6phosphate is catalyzed by fructose26bisphosphatase H20 FBPaseZ These two catalytic activities are contained in a single protein compleX referred to as a quotbifunctionalquot protein PFKZ is activated by dephosphorylation of the compleX while FBPaseZ is activated by phosphorylation of the compleX F i catalyzed by protein kinase A fructose26bisphosphate Bifunctional protein phosphorylated FBPase Z active PFK 2 inactive fructose6phosphate Glycolysisgluconeogenesis 27 fructose6phosphate ATP f1uctse6phosphate fructose phosphate P ATP PFK Z active l ADP inactivation activation X PFK1 fructose 26 bisphosphate O I FBPasel gluconeogenesis glycolysis fructose16bisphosphate fructose 16 bisphosphate ADP Figure 5 Formation of fructose26bisphosphate in the fed state leads to activation of phosphofructokinasel in glycolysis and inactivation of gluconeogenesis PFKl phosphofructokinasel PFKZ phosphofructokinaseZ FBPasel fructose16bisphosphatase Link to hormone signals In the fed state insulin is released from the Bcells of the pancreas in response to dietary glucose in the circulation Insulin activates a protein phosphatase that removes phosphate from regulated proteins One of the substrates for protein phosphatase is the bifunctional protein for the metabolism of fructose 26bisphosphate As noted above in this instance only phosphofructokinaseZ is active Thus in the presence of insulin in the fed state fructose26bisphosphate is produced so that in the liver it can serve as a signal of elevated blood glucose Fig 5 Fructose26bisphosphate in turn activates liver glycolysis to process the carbohydrate from the diet Concurrently gluconeogenesis is shut off inactivated directly by the action of fructose26bisphosphate on fructose16bisphosphatase because there is no need to synthesize glucose after a carbohydrate meal Allasteric Regulation onymvute Kinase in Liver Besides PFKl regulation of liver glycolysis occurs at pyruvate kinase thus providing coordination within glycolysis and between glycolysis and gluconeogenesis Coordination within glycolysis is accomplished by both citrate and ATP acting as allosteric inhibitors of the active form of pyruvate kinase just as both of these regulators allosterically inhibit PFKl Inhibition of liver glycolysis is also coordinated with the activation of gluconeogenesis both through allosteric control and covalent modi cation phosphorylation Alanine is the primary amino acid precursor for the synthesis of glucose and its concentration in the blood rises dramatically during starvation Alanine shuts off glycolysis at pyruvate kinase by allosterically inhibiting the active form of the enzyme Fig 7 Thus during gluconeogenesis this action of alanine ensures that phosphoenolpyruvate is kept in the gluconeogenic pathway to be processed to glucose instead of immediately being converted to pyruvate Activity of the liver pyruvate kinase decreases in response to glucagon following phosphorylation of the enzyme by protein kinase A Fig 6 Direct phosphorylation of hepatic pyruvate kinase causes it to become inactive Glucagon as a signal of starvation coordinates a decreased activity of glycolysis by causing the inhibition of both phosphofructokinasel due to the disappearance of fructose26bisphosphate and pyruvate kinase directly by phosphorylation At the same time the activity of gluconeogenesis is increased through activation of fructose16 Glycolysisgluconeogenesis 28 bisphosphatase due to the disappearance of fructose26bisphosphate In the fed state insulin activates protein phosphatase to reverse the effects initiated by glucagon both on pyruvate kinase and on the metabolism of fructose26bisphosphate glucagon protein kinase A Pyruvate ATP OPO3 39 Pyruvate curate PquVate Q ATP kinase Km se alanine actlve inactive Pi Phosphoenolpyruvate ADP protein phosphatase insulin GD Figure 6 Regulation of liver pyruvate kinase The bold faced enzyme is active The italicized enzyme is inactive due to phosphorylation of the protein In response to low blood glucose glucagon stimulates production of cyclic AMP which in turn activates protein kinase A Protein kinase A then phosphorylates pyruvate kinase leading to this inactivation Under high glucose conditions insulin activates protein phosphatase to dephosphorylate the enzyme thus reversing the effects of phosphorylation leading to activation Allosteric inhibitors of the active dephosphorylated form are shown Glycolysisgluconeogenesis 29
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