Carbohydrate Metabolism 2: Glycogen degradation, glycogen synthesis, reciprocal regulation of glycogen metabolism Bioc 460 Spring Lecture 34 (Miesfeld) Gerty Cori won the 1947 Nobel Prize for her work on glycogen metabolism Carbohydrates in pasta are a good way to replenish muscle glycogen stores Glycogen phosphorylase enzyme is a dimer that is regulated by both phosphorylation and allostery

Glycogen is a highly-branched polymer of glucose that can be quickly degraded to yield glucose-1P which is isomerized to glucose-6P. Glycogen phosphorylase removes one glucose at a time from the nonreducing ends using inorganic phosphate (P i ). Glycogen synthase adds glucose residues to nonreducing ends in a reaction involving UDP-glucose; the cost of glycogen synthesis is 1 ATP/glucose residue. Net phosphorylation leads to glycogen degradation, whereas, net dephosphoryation, results in glycogen synthesis. Key Concepts in Glycogen Metabolism

Overview of Glycogen Metabolism The storage form of glucose in most eukaryotic cells (except plants) is glycogen, a large highly branched polysaccharide consisting of glucose units joined by  - 1,4 and  -1,6 glycosidic bonds. 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.

The reducing and nonreducing ends of glycogen The nonreducing end of glycogen molecules refers to the carbon that is opposite to the reducing end in the ring structure. The reducing end of a linear glucose molecule can be oxidized by Cu 2+ by definition. Reducing end Nonreducing end ……… Reducing end

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. Glycogen is stored primarily in liver and skeletal muscle cells.

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 + P i → 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 + 2P i Pathway Questions

3. What are the key enzymes in glycogen metabolism? Glycogen phosphorylase – enzyme catalyzing the phosphorylysis reaction that uses P i to remove one glucose at a time from nonreducing ends of glycogen resulting in the formation of glucose-1P.. Glycogen synthase - enzyme catalyzing the addition of glucose residues to nonreducing ends of glycogen using UDP-glucose as the glucose donor. Branching and debranching enzymes - these two enzymes are responsible for adding (branching) and removing (debranching) glucose residues.

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. Key is to deplete glycogen before carbo loading to get 2x glycogen level.

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 (P i ) 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 (ΔGº' = +3.1 kJ/mol), making the reaction unfavorable, the actual change in free energy is favorable (ΔG' = -6 kJ/mol) due to the high concentration of P i 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 P i substrate is bound to the active site by interactions with pyridoxal phosphate and active site amino acids.

Function of Phosphoglucomutase The the next reaction in the glycogen degradation pathway is the conversion of glucose-1P to glucose-6P by the enzyme phosphoglucomutase. Where have you seen this type of reaction before (a mutase rxn)?

Glycogen Debranching Enzyme 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. 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 glucose-1P and glucose?

Regulation of Glycogen Phosphorylase Activity 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 phosphorylase a (active), and the unphosphorylated form is called phosphorylase b. It is the same polypeptide, just a different name.

The enzyme responsible for phosphorylating glycogen phosphorylase b to activate it, is phosphorylase kinase which is a downstream target of glucagon and epinephrine signaling, as well as, insulin signaling. Regulation of Glycogen Phosphorylase Activity

Tissue-specific isozymes of glycogen phosphorylase The activity of glycogen phosphorylase can also be controlled by allosteric regulators, which binds to the T state and shifts the equilibrium to the R state. Liver and muscle isozymes of glycogen phosphorylase are allosterically-regulated in different ways, which reflects the unique functions glycogen in these two tissues.

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. 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? Hint: what does the liver do with the glucose-6P that is produced? Tissue-specific isozymes of glycogen phosphorylase

Glycogen synthase catalyzes glycogen synthesis 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). The rapid hydrolysis of PP i by the abundant cellular enzyme pyrophosphatase results in a highly favorable coupled reaction. Why does rapid conversion of PP i --> 2 P i result in a more favorable reaction?

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 using a α-1,6 branchpoint.

Growing Glycogen Tree - Starting with Glycogenin Protein

Regulation of Glycogen Synthase Activity The activity of glycogen synthase is also primarily controlled by reversible phosphorylation. Dephosphorylation activates glycogen synthase, whereas, glycogen phosphorylase is activated by phosphorylation. In this case, the active glycogen synthase a (active) form is dephosphorylated and favors the R state, whereas, the inactive glycogen synthase b form is phosphorylated and favors the T state. The “a” form is always the active form; glycogen phosphorylase “a” is phosphorylated, whereas, glycogen synthase “a” is dephosphorylated.

Hormone activation of glycogen synthase activity is mediated by insulin, which promotes the activation of glycogen synthase by stimulating PP-1 activity. Epinephrine and glucagon signaling leads to inactivation of glycogen synthase. Regulation of Glycogen Synthase Activity

Reciprocal regulation of glycogen metabolism Since glycogen phosphorylase and glycogen synthase have opposing effects on glycogen metabolism, it is critical that their activities be reciprocally regulated to avoid futile cycling and to efficiently control glucose-6P concentrations within the cell. What is the metabolic logic of glucose inhibition of glycogen phosphorylase activity and activation of glycogen synthase?

Net phosphorylation drives glycogen degradation, and net dephosphorylation drives glycogen synthesis. Hormone signaling in liver cells

cAMP triggers two types of phosphorylation circuits in muscle cells; one that stimulates glycogen degradation and a second that inhibits glycogen synthesis. Glucagon signaling

Insulin signaling results in dephosphorylation of glycogen metabolizing enzymes and elevated rates of glycogen synthesis. Insulin signaling

Human glycogen storage diseases A number of human diseases have been identified that affect glycogen metabolism. Disease symptoms in many cases include: liver dysfunction due to excess glycogen fasting- induced hypoglycemia (low blood glucose levels) in the most severe diseases, death at an early age.

Human glycogen storage diseases von Gierke's disease is due to a deficiency in the enzyme glucose-6- phosphatase which causes a build-up of glycogen in the liver because glucose- 6P accumulates and activates glycogen synthase. McArdle's disease harbor defects in muscle glycogen phosphorylase. These individuals suffer from exercise-induced muscle pain due to their inability to degrade muscle glycogen.