Class Note for BIOC 460 at UA 3
Class Note for BIOC 460 at UA 3
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Date Created: 02/06/15
Biochemistry 460 Dr Tischler GLYCOGEN ALLOSTERIC CONTROL Related Reading Chapter 21 592601 604607 611612 in Stryer 639h edition OBJECTIVES Glycogenolysis pathway 1 Compare and explain the different fates of glucose6phosphate from glycogenolysis in liver and in muscle 2 Distinguish between the tense and relaxed forms of glycogen phosphorylase in terms of activity of this enzyme 3 Describe the function of phosphorylase kinase in promoting the mobilization of glycogen relative to the phosphorylation state of glycogen phosphorylase 4 Explain the physiological basis ie rationale of the effect for AMP allosterically activating glycogen phosphorylase 5 Explain the physiological basis ie rationale of the effect for calcium activating phosphorylase kinase Glycogenesis pathway 1 Explain the physiological basis ie rationale of the effect for glucose6phosphate allosterically activating glycogen synthase 2 Describe the differences between the two forms of glycogen synthase ie dform and iform PHYSIOLOGICAL PREMISE Muscle is remarkably well adapted metabolically to handle activities from a short duration lift of a heavy load to running a marathon During the rst few seconds of initiating muscle activity creatine phosphate provides energy to immediately maintain the amount of ATP in the cell This instantaneous source of energy buys you the time to begin breaking down glycogen as a source of energy Fortuitously muscle does not contain glucose6phosphatase so that instead of making glucose muscle breaks down glycogen to provide fuel just for that cell The large glycogen stores in bulky muscles enable the athlete to sprint using anaerobic glycolysis while the longdistance runner relies on aerobic metabolism In adapting to longterm exercise the body diverts blood towards the aerobic skeletal muscles which largely use fats as an energy source for hours Failure to make the switch from glycogen to fats is termed quothitting the wallquot since glycogen cannot sustain muscle energy levels for extended periods of time Glycogen Allosteric Control l GLYCOGEN STRUCTURE AND GENERAL ASPECTS Glycogen is the storage form of glucose Liver and kidney store glycogen that can provide 75 of the glucose needed mostly by red blood cells and the brain during the rst 24 hours of food deprivation Muscle stores glycogen solely for its own needs The lack of glucose6 phosphatase in muscle prevents formation and secretion of glucose by muscle for use by other tissues Glycogen has a highly branched structure to increase its solubility and to provide many more sites for removal of glucose units that are phosphorylated by glycogen phosphorylase see below The glucose residues in the linear chain are joined by ocl4 glycosidic bonds and branches are formed by creation of ocl6 glycosidic bonds Fig l H20H H20H L 041 6 linka F 1 Th ge lgure e O glycogen structure CHZOH CHon EH2 CHZOH showing the glycosidic bonds L between the hydroxyl groups on carbons 4 linear chain or 6 branch point of one glucose residue and 0c14 linkages carbon 1 of another residue PATHWAYS OF GLYCOGEN METABOLISM Degradation of glycogen glycogenolysis Glycogen phosphorylase is the principal enzyme of glycogenolysis that catalyzes the cleavage and phosphorylation of glucose residues at the ends of branches see Fig l for glycogen structure This process is a phosphorolysis involving addition of orthophosphate across the glycosidic bond with the release of glucoselphosphate as a product The overall reaction is glycogen 11 glucose residues inorganic phosphate Pi glucoselphosphate glycogen n I residues Besides binding sites for its substrates glycogen and Pi glycogen phosphorylase contains a prosthetic group cofactor pyridoxal phosphate that is derived from vitamin B6 pyridoxine Additionally glycogen phosphorylase contains allosteric binding sites for AMP an activator and allosteric inhibitors including ATP and glucose6phosphate The enzyme can also be activated by covalent modi cation via phosphorylation in response to hormonal signals and thus eXists in a phosphorylated a form and a non phosphorylated b form Glycogen phosphorylase removes all glucose residues up to four sugars from a branch point The combined actions of a debranching enzyme and a transferase move three of the four remaining glucose residues of a branch to the end of another branch so that they can be removed by glycogen phosphorylase The fourth glucose residue of the branch is cleaved by a glucosidase that produces free glucose as a product Depending on the tissue this free glucose may be released to the blood liver or phosphorylated by hexokinase to glucose6phosphate muscle The tissue where the glycogen is stored liver or muscle determines the ultimate fate of the glucoselphosphate product In both instances the product is converted to glucose6phosphate via phosphoglucomutase Fig 2 Glycogen Allosteric Control 2 Liver glycogenalysis Glycogen glycogen Pi h h p 539 ry ase LIVER PATHWAY GIucose1P phosphoglucoml tase glucose6phosphatase Glucose6P GLUCOSE glycolysis inhibited by lack of F26BP Figure 2a Glycogenolysis and the fate of glycogen in liver and kidney F26BP is fructose26 bisphosphate Glycogen Pi glycogen phosphorylase MUSCLE PATHWAY GIucose1P phosphoglucomutase l GIucose6P glycolysis activated by AMP anaerobic glycolysis when energy low Pyruvate LaCtate pyruvate lactate dehydrogenase dehvdroclenase Acetyl CoA C02 aerobic metabolism Figure 2b Glycogenolysis and the fate of glycogen in muscle Glycogen Allosteric Control 3 During the postabsorptive nutritional state the glycogen stored in liver is needed to sustain blood glucose levels once dietary glucose has been consumed Consequently phosphate is removed from the glucose6 phosphate to form glucose via glucose phosphatase Fig 2a This enzyme is also the last step of the gluconeogenic pathway as discussed in an earlier lecture In these organs the glucose6phosphate is not metabolized via glycolysis because that pathway is inhibited at the phosphofructokinasel reaction Following food deprivation the concentration of fructose26bisphosphate in the liver decreases causing a loss of activity of phosphofructokinasel Thus in liver activation of glycogenolysis coincides with inhibition of glycolysis The hormonal events primarily responsible for this coordinated regulation will be considered in the next lecture Muscle glycogenolysis Other tissues such as muscle lack glucose6phosphatase and cannot synthesize glucose Consequently when muscle or heart degrades glycogen it is for the sole purpose of providing energy for the cell in which the glycogen was stored For instance during exercise glycogen phosphorylase and phosphofructokinasel are active concurrently so that the glucose6phosphate derived from glycogenolysis can be used by glycolysis for energy production Fig 2b In relatively anaerobic muscles the pyruvate from glycolysis is reduced to lactate see glycolysis lecture whereas under aerobic conditions pyruvate is oxidized completely to carbon dioxide Synthesis of glycogen glycogenesis Glucose Hexokin ase ATP muscle Glucokinase ADP Glucosen Glucosen1 liver GIucose6phosphate Phosphoglucomutase Glucose1P Uriclvltransferase GIucose1phosphate UDPglucose UDP Glycogen Synthase UTP PR gt 2Pi Figure 3 Pathway of glycogen synthesis glycogenesis Glucose6phosphate is derived from glucose via hexokinase in muscle or glucokinase in liver Glycogen is usually synthesized from glucose Fig 3 Glucose is phosphorylated in glycolysis by glucokinase liver or hexokinase muscle kidney to produce glucose6phosphate The glucose6 phosphate is then converted to glucoselphosphate by phosphoglucomutase the reverse of the reaction used during production of glucose6phosphate from glycogen The glucose residue to be added to the growing glycogen molecule must rst be chemically activated by attachment of uridine monophosphate UMP to glucoselphosphate to form UDPglucose The UMP is derived from UTP and is attached in a reaction catalyzed by glucoselphosphate uridyltransferase This transferase is highly exergonic very negative AG by virtue of the release of pyrophosphate that hydrolyzes readily to two molecules of inorganic phosphate with an energy yield greater than 13 kcalmol Glycogen Allosteric Control 4 UDPglucose is the substrate for glycogen synthase the principal and regulated enzyme of glycogenesis Glycogen synthase is regulated by covalent modi cation phosphorylation in response to hormonal signals and by allosteric activation These regulatory mechanisms will be discussed later The formation of branches is catalyzed by a branching enzyme that moves 7 glucose residues from the end of a growing chain to an interior area where a branch can be attached ALLOSTERIC REGULATION OF GLYCOGEN METABOLISM The regulation of glycogen synthase glycogenesis and glycogen phosphorylase glycogenolysis opposes each other Because synthesis of glycogen requires energy while glycogenolysis does not directly produce energy it would be deleterious for the cell to have both pathways operating simultaneously Both enzymes are regulated by hormonal control through phosphorylation dephosphorylation reactions The hormonal mechanism will be discussed in depth in the next lecture A general concept to remember is that phosphorylation of enzymes is associated with physiological responses that require recruitment of fuel eg starvation stress ght or ight response Hence phosphorylation activates those enzymes responsible for mobilizing the fuels eg glycogen fats while in parallel phosphorylation inactivates those enzymes linked to fuel synthesis and storage Conversely dephosphorylation of enzymes is associated with physiological conditions in which fuel is stored ie fed state and the activation of enzymes will follow a pattern opposite to that for fuel mobilization Glycogen Phosphorylase The regulation of glycogen phosphorylase is complex Fig 4 Regulation ensures that in liver glucose remains stored as glycogen until it is mobilized for maintaining blood glucose homeostasis In muscle glycogen remains stored until it is mobilized to supply energy to the cell The active form of the enzyme is termed quotrelaxedquot while the inactive form is termed quottensequot Phosphorylation of glycogen phosphorylase causes the enzyme to revert spontaneously to its relaxed state Dephosphorylation to the tense state is catalyzed by protein phosphatase 1 Fig 4 The phosphorylated form is designated as phosphorylasea and the dephosphorylated form is designated as phosphorylaseb The enzyme that directly catalyzes the phosphorylation of glycogen phosphorylase is phosphorylase kinase Phosphorylase kinase can be activated allosterically by calcium In muscle calcium is released from the sarcoplasmic reticulum in response to nerve signals associated with muscle contraction Thus during muscle contraction the release of calcium is followed by activation of phosphorylase kinase that in turn catalyzes the phosphorylation of glycogen phosphorylase to its relaxed active form Additionally phosphorylase kinase is activated in response to a variety of hormone signals in a cascade that leads to activation of glycogenolysis to be discussed in the next lecture Allosteric control of glycogen phosphorylase also causes interconversion between its relaxed and tense states AMP is generated in the cytoplasm of muscle cells under conditions in which cell energy is depleted such as following intense or prolonged exercise Thus AMP in the cytoplasm provides a signal that energy is low in the cell as opposed to a high concentration of ATP re ecting a state of energy abundance The binding of AMP to its allosteric site on glycogen phosphorylase promotes the conversion of the tense inactive form of phosphorylaseb to the relaxed active state of phosphorylaseb Fig 4 By AMP activating glycogen phosphorylase glycogen can be mobilized to meet the energy needs of the cell Recall that AMP also activates phosphofructokinasel in glycolysis so that in muscle glycogenolysis and glycolysis are turned on concurrently see Fig 2b Conversely a high level of ATP the signal of abundant energy maintains phosphorylaseb in its tense inactive state Fig 4 Glycogen Allosteric Control 5 activation by calcium in muscle inhibition by glucose6P I phosphorylaseb phosphorylasea OH ATP v ADP phosphorylase OPOs kinase tense inactive protein phosphatase1 glucose6P muscle or liver AMP OH ATP muscle muscle OPO3 relaxed active Figure 4 Regulation of glycogen phosphorylase The larger thicker arrows represent preferred directions Glycogen phosphorylase exists in a dephosphorylated b and phosphorylated a state Phosphorylase kinase catalyzes the phosphorylation and it is allosterically regulated The active form of glycogen phosphorylase is the relaxed state that can be achieved by spontaneous activation following phosphorylation or by allosteric activation of the b form in muscle by AMP Allosteric conversion to the tense form is promoted by ATP or glucose6P in muscle Glucose6phosphate also causes inactivation of glycogen phosphorylase The physiological basis for this inhibitory effect of glucose6phosphate relates to the fact that accumulation of excessive amounts of glucose6phosphate would deplete the cell of inorganic phosphate that is required for the synthesis of ATP Hence it is important to the cell that the concentration of glucose6phosphate be maintained at a low level by inhibiting glycogenolysis and by increasing the formation of glycogen see glycogen synthase below Elevated levels of glucose6phosphate maintain phosphorylaseb in its tense inactive conformation in two ways First glucose6phosphate binds directly to glycogen phosphorylase as an allosteric inhibitor to promote conversion from the relaxed to the tense conformation Second glucose6 phosphate allosterically inhibits phosphorylase kinase to prevent phosphorylation of the enzyme to phosphorylasea the other relaxed form of the enzyme Glycogen Allosteric Control 6 Glycogen Synthuse Like glycogen phosphorylase glycogen synthase also exists in phosphorylated and dephosphorylated forms Fig 5 This enzyme is responsible for fuel synthesis and storage It is active when dephosphorylated and inactive when phosphorylated opposite to that of glycogen phosphorylase A variety of protein kinases including phosphorylase kinase can catalyze the phosphorylation of glycogen synthase Protein phosphatasel catalyzes the dephosphorylation of the enzyme just as it does for glycogen phosphorylase The phosphorylated form of the enzyme is designated as glycogen synthased The quotdquot indicates that the phosphorylated form depends on glucose6phosphate being bound to the enzyme for activity This effect like the effect of glucose6phosphate on glycogen phosphorylase described above provides a mechanism for ensuring that glucose6phosphate does not accumulate and decrease the cell s ability to make ATP by depleting phosphate stores Also it provides opposite control of glycogen metabolism by activating the synthase and inhibiting the phosphorylase Note that the activity of the dephosphorylated form of glycogen synthase is independent glycogen synthasei of glucose6phosphate for its activity since dephosphorylation places the enzyme in a favorable conformation to carry out its catalytic role variety of GI protein kinases ycogen synthasei ATP ADP Glycogen I synthased OH OPO gucose6 P active 7 3 p enzyme Pi active protein inactive phosphatase Figure 5 Regulation of glycogen synthase Note that glycogen synthase is inactive when glycogen phosphorylase is active This reciprocal control prevents energy wasting GLYCOGEN STORAGE DISEASES The defect in Type I von Gierke s disease is glucose6phosphatase This defect impairs glucose production both from glycogenolysis and from gluconeogenesis because the defective enzyme is common to both pathways Because gluconeogenesis is also impaired patients eXhibit lactic acidemia which is excessive accumulation of lactic acid in blood that can lower the blood pH These patients must eat frequent meals to prevent severe hypoglycemia Even a few hours of food deprivation is problematic because once dietary glucose is gone blood levels of glucose will decline quickly as red blood cells and brain tissue continue using glucose Patients with von Gierke s disease may have enlarged livers hepatomegaly because mobilization of stored glycogen is limited Glycogen Allosteric Control 7 The Type V disease McArdle s is caused by a defect of glycogen phosphorylase in skeletal muscle This does not lead to hypoglycemia because muscle does not use glycogen to produce glucose Instead McArdle s disease severely impairs strenuous exercise since the patient cannot mobilize glycogen as an initial fuel for the muscle until fatty acids become available Individuals with McArdle s disease exhibit little production of lactate during strenuous exercise because the breakdown of glycogen is minimal Because these patients cannot rely on glycogen as a source of energy during strenuous exercise their muscles are completely depleted of creatine phosphate when patients attempt to do so In normal individuals creatine phosphate provides energy for a few seconds to buy time until glycogenolysis is initiated to provide energy Type VI disease is also caused by a defect in glycogen phosphorylase but this is speci c to liver It is possible to have separate defects of glycogen phosphorylase is muscle and liver because the glycogen phosphorylase enzymes in these two tissues are encoded on different genes Like Type I Type VI is also characterized by hypoglycemia and by hepatomegaly An important difference is that in Type VI the gluconeogenesis pathway is functional and lactic acidemia is not observed Medical Scenario SS a 9monthold female suffers from hypoglycemia and hepatomegaly She was breast fed for 6 months after which frequent feedings were given Examination shows a stubby baby in the 3ml percentile for height and weight The liver is enlarged and can be felt 5 inches below the ribs 9nomrally should not be felt Additional blood analyses are provided in the table below Her brothers aged 9 brother 1 and 10 brother 2 had shown similar symptoms and are being treated with corn starch enriched feedings to which oral bicarbonate is added Their current blood analyses are also included in the table Measurement Patient Brother 1 Brother 2 Normal Cholesterol 430 mgdL 147 135 lt200 Triglycerides 1832 mgdL 175 154 lt175 Uric acid 12 mgdL 67 70 3 to 7 Lactate 38 mmolL 27 28 05 to 30 A liver biopsy of the patient showed reduced activity of glucose6phosphatase PCR analysis revealed a GtoT transition leading to a glymaval alteration Glycogen Allosteric Control 8
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