Lecture 34. Carbohydrate Metabolism 2. Glycogen. Key Concepts. Biochemistry and regulation of glycogen degradation

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1 Lecture 34 Carbohydrate Metabolism 2 Glycogen Key Concepts Overview of Glycogen Metabolism Biochemistry and regulation of glycogen degradation Biochemistry and regulation of glycogen synthesis What mechanisms regulate the activity of muscle and liver glycogen phosphorylase?

2 Three primary pathways in anabolic carbohydrate metabolism in nonphotosynthetic organisms: 1.pentose phosphate pathway 2.gluconeogenesis 3.glycogen metabolism Metabolism of ribose sugars in the pentose phosphate pathway is used to generate NADPH and to provide the carbohydrate component of nucleotides The major sources of carbon in gluconeogenesis are amino acids and glycerol in animals, and glyceraldehyde-3- phosphate (GAP) in plants. Overview of Glycogen Metabolism Glycogen the storage form of glucose in most eukaryotic cells (except plants) a large highly branched polysaccharide consisting of glucose units joined by α-1,4 and α-1,6 glycosidic bonds Glycogen degradation and synthesis occurs in the cytosol substrate for these reactions is the free ends of the branched polymer (nonreducing ends) The large number of branch points in glycogen results in the generation of multiple nonreducing ends that provide a highly efficient mechanism to quickly release and store glucose.

3 Overview of the Glycogen Metabolism The large number of branch points in glycogen results in the generation of multiple nonreducing ends that provide a highly efficient mechanism to quickly release and store glucose. Glycogen Core Complexes Glycogen core complexes consist of glycogenin protein and ~50,000 glucose molecules with α- 1,6 branches about every 10 residues creating ~2,000 nonreducing ends. Twenty to forty glycogen core complexes associate inside liver and muscle cells to form glycogen particles containing over a million glucose molecules. These glycogen particles can be visualized by electron microscopy and account for up to 10% by weight of liver tissue.

4 Pathway Questions 1. What purpose does glycogen metabolism serve in animals? Liver glycogen is used as a short term energy source for the organism by providing a means to store and release glucose in response to blood glucose levels; liver cells do not use this glucose for their own energy needs. Muscle glycogen provides a readily available source of glucose during exercise to support anaerobic and aerobic energy conversion pathways within muscle cells; muscle cells lack the enzyme glucose-6-phosphatase and therefore cannot release glucose into the blood. Pathway Questions 2. What are the net reactions of glycogen degradation and synthesis? Glycogen Degradation: Glycogen n units of glucose + Pi Glycogen n-1 units of glucose + glucose-6-phosphate Glycogen Synthesis: Glycogen n units of glucose + glucose-6-phosphate + ATP + H 2 O Glycogen n+1 units of glucose + ADP + 2Pi

5 Pathway Questions 3. What are the key enzymes in glycogen metabolism? Glycogen phosphorylase enzyme catalyzing the phosphorylysis reaction that uses Pi to remove one glucose at a time from nonreducing ends of glycogen resulting in the formation of glucose-1p. Liver and muscle glycogen phosphorylase are isozymes (two different genes) that are both activated by phosphorylation but have distinct responses to allosteric effectors. Glycogen synthase - enzyme catalyzing the addition of glucose residues to nonreducing ends of glycogen using UDP-glucose as the glucose donor. Glycogen synthase activity is inhibited by phosphorylation; binding of the allosteric activators glucose or glucose-6p promotes dephosphorylation and enzyme activation. Branching and debranching enzymes - these two enzymes are responsible for adding (branching) and removing (debranching) glucose residues to the glycogen complex through the cleavage and formation of α-1,6 glycosidic bonds. Pathway Questions 4. What are examples of glycogen metabolism in real life? The performance of elite endurance athletes can benefit from a diet regimen of carbohydrate "loading" prior to competition. Carbohydrate loading regimens can result in a build-up of stored muscle glycogen that is sometimes higher than what can be obtained by simply following a high carbohydrate diet.

6 Biochemistry of Glycogen Degradation Overview of the Glycogen Metabolism The large number of branch points in glycogen results in the generation of multiple nonreducing ends that provide a highly efficient mechanism to quickly release and store glucose.

7 Function of Glycogen Phosphorylase Glycogen degradation is initiated by glycogen phosphorylase, a homodimer that catalyzes a phosphorolysis cleavage reaction of the α-1,4 glycosidic bond at the nonreducing ends of the glycogen molecule. Inorganic phosphate (Pi) attacks the glycosidic oxygen using an acid catalysis mechanism that releases glucose-1p as the product. Although the standard free energy change for this phosphorylysis reaction is positive ( Gº' = +3.1 kj/mol), making the reaction unfavorable, the actual change in free energy is favorable ( G' = -6 kj/mol) due to the high concentration of Pi relative to glucose-1p inside the cell (ratio of close to 100). Structure of Glycogen Phosphorylase Exists as a dimer and has binding sites for glycogen and catalytic sites that contain pyridoxal phosphate (derived from vitamin B6). The critical Pi substrate is bound to the active site by interactions with pyridoxal phosphate and active site amino acids.

8 Function of Phosphoglucomutase The glucose-1p product of the glycogen phosphorylase reaction is not an intermediate in glycolysis and liver cells do not contain a glucose-1p-phosphatase. Therefore, the next reaction in the glycogen degradation pathway is the conversion of glucose-1p to glucose-6p by the enzyme phosphoglucomutase. The enzyme first donates a phosphate group to the substrate to generate an intermediate bisphosphate compound, and then the bisphosphate compound is dephosphorylated to regenerate the phosphoenzyme and release the product. Fate of glucose-6-phosphate differs by cell type In liver cells, glucose-6p is dephosphorylated by glucose-6- phosphatase to generate glucose for export. In muscle cells which lack glucose-6-phosphatase, the glucose-6p is used as a source of chemical energy in glycolysis.

9 Overview of the Glycogen Metabolism The large number of branch points in glycogen results in the generation of multiple nonreducing ends that provide a highly efficient mechanism to quickly release and store glucose. Glycogen Debranching Enzyme Glycogen phosphorylase removes glucose units from the nonreducing end until it reaches within four glucose units of an α-1,6 branchpoint. The glycogen debranching enzyme (also called α-1,6-glucosidase) recognizes the partially degraded branch structure and remodels the substrate in a two step reaction. 1) the debranching enzyme transfers three glucose units to the nearest nonreducing end to generate a new substrate for glycogen phosphorylase. 2) the bifunctional debranching enzyme cleaves the α-1,6 glycosidic bond to release free glucose. Since α-1,6 branch points occur about once every 10 glucose residues in glycogen, complete degradation releases ~90% glucose-1p and 10% glucose molecules. Is there a difference in the amount of energy that can be recovered from these two molecules (glucose-1p and glucose)?

10 Glycogen Debranching Enzyme Regulation of Glycogen Degradation

11 Regulation of Glycogen Phosphorylase Activity is regulated by both covalent modification (phosphorylation) and by allosteric control (energy charge). Glycogen phosphorylase is found in cells in two conformations: active conformation, R form inactive conformation, T form Phosphorylation of serine 14 (Ser 14) shifts the equilibrium in favor of the active R state. This phosphorylated form of glycogen phosphorylase is called the a form, or simply phosphorylase a, and the unphosphorylated form is the b form, or phosphorylase b. Phosphorylase Kinase and Protein Phosphatase-1 The enzyme responsible for phosphorylating glycogen phosphorylase b is phosphorylase kinase which is a downstream target of glucagon and epinephrine signaling. Insulin signaling stimulates the activity of protein phosphatase-1 (PP-1) leading to inactivation of glycogen phosphorylase. Protein phosphatase 1 is the same insulin-regulated enzyme that dephosphorylates PFK-2/FBPase-2, the enzyme responsible for controlling fructose-2,6-bp levels.

12 Different isozymes of glycogen phosphorylase The activity of glycogen phosphorylase can also be controlled by allosteric regulators, which bind to the enzyme and shift the equilibrium. Liver and muscle isozymes of glycogen phosphorylase are allosterically-regulated in different ways, which reflects the unique functions glycogen in these two tissues Muscle glycogen phosphorylase b but not liver glycogen phosphorylase b can be shifted from the T to R state by binding of the allosteric activator AMP. ATP and glucose-6p function as allosteric inhibitors of muscle glycogen phosphorylase b.

13 Liver glycogen phosphorylase a, but not muscle glycogen phosphorylase a is subject to allosteric inhibition by glucose binding which shifts the equilibrium from the R to T state. When liver glycogen phosphorylase a (phosphorylated form) is shifted to the T state, it is a better substrate for dephosphorylation by PP-1 than is the R state. Regulation of Glycogen Degradation question: Why does it make sense that muscle glycogen phosphorylase b, but not liver glycogen phosphorylase b, would be allosterically activated by AMP in the absence of hormone signaling?

14 Biochemistry and Regulation of Glycogen Synthesis Meet UDP-Glucose The addition of glucose units to the nonreducing ends of glycogen by the enzyme glycogen synthase requires the synthesis of an activated form of glucose called uridine diphosphate glucose (UDP-glucose). Glucose-6P is first converted to glucose-1p by phosphoglucomutase, and then the enzyme UDP-glucose pyrophosphorylase catalyzes a reaction involving the attack of a phosphoryl oxygen from glucose-1p on the gamma phosphate of uridine triphosphate (UTP). The rapid hydrolysis of PPi by the abundant cellular enzyme pyrophosphatase results in a highly favorable coupled reaction.

15 Glycogen Synthase Reaction Glycogen synthase transfers the glucose unit of UDP-glucose to the C-4 carbon of the terminal glucose at the nonreducing end of a glycogen chain. The UDP moiety is released and UTP is regenerated in a reaction involving ATP and the enzyme nucleoside diphosphate kinase. Glycogen Branching Enzyme Once the chain reaches a length of 11 glucose residues, the glycogen branching enzyme transfers seven glucose units from the end of the chain to an internal position by creating a new α-1,6 branchpoint. This new branchpoint must be at least four glucose residues away from the nearest branch. Not the exact reverse of the debranching enzyme s reaction!

16 Growing Glycogen Tree Glycogen Synthase Regulation The activity of glycogen synthase is also primarily controlled by reversible phosphorylation. The effect of phosphorylation on the activity of glycogen synthase is however the reverse of what we saw with glycogen phosphorylase. Dephosphorylation activates glycogen synthase, whereas, glycogen phosphorylase is activated by phosphorylation. In this case, the active glycogen synthase a form is dephosphorylated and favors the R state, whereas, the inactive glycogen synthase b form is phosphorylated and favors the T state.

17 Hormones control Glycogen Synthase Regulation Hormone activation of glycogen synthase activity is mediated by insulin, which promotes the dephosphorylation and activation of glycogen synthase by stimulating PP-1 activity. Epinephrine and glucagon signaling results in phosphorylation and inactivation of glycogen synthase. These kinases include protein kinase A (PKA), protein kinase C (PKC), glycogen synthase kinase 3 (GSK3) and calmodulin dependent kinase (CAMK). GSK3 phosphorylation of glycogen synthase requires that it first be phosphorylated by CAMK, which functions as a priming event.