Glycogen Metabolism OUTLINE • Glycogen breakdown requires the interplay of several enzymes • Phosphorylase is regulated by allosteric interactions and reversible phosphorylation • Epinephrine and glucagon signal the need for glycogen breakdown • Glycogen is synthesized and degraded by different pathways • Glycogen breakdown and synthesis are reciprocally regulated Glycogen synthesis: glycogenesis Degradation of glycogen: glycogenolysis.
Glycogen Glycogen is a highly branched, very large polymer of glyc mols linked 1 4 Branches arise by 1 6 at about every 8-10th residue It is found in the cytosol. It is the storage form of Glc. Liver and muscle are the major sites for the storage of glycogen.
Surface: nonreducing endsAt the core:glycogenin Degradation: takes place at the surface!
Degradation of Glycogen Is not a simple reversal of the synthetic pathway Other enzymes involved Glycogen G-1-P Shortening of chains: – Glycogen phosphorylase ( -1 4) – It is an exoglucosidase – Degrades the gly. chains at their non-reducing ends until four glucosyl units remain on each chain before the branch point – The resulting structure a limit dextrin – Phosphorylase cannot degrade this any further!
Degradation of Glycogen continuedRemoval of branches: Branches are removed through two enzymatic activities of the debranching enzyme a. Glucosyl 4:4 transferase removes the outer 3 of 4 glucosyl residues b. Single glucose residue attached in an 16 linkage is then removed by the -amylo (1:6) glucosidase activity of the debranching enzyme, releasing free glucose
Regulation phosphorylaseRegulation of glycogen metabolism is different in muscleand liver. In muscle, the end served by glycolysis is ATP production and the rate of glycolysis increases as muscle works more, demanding more ATP. The liver has a different role in whole-body metabolism and glucose metabolism in the liver is different. The liver makes sure that glucose level is constant in the blood, producing and exporting Glc.
Regulation of Glycogen BreakdownGlycogen represents the most immediately available large-scalesource of metabolic energy. Therefore, it is important thatanimals be able to activate glycogen mobilization very rapidly. Glycogen breakdown is an hormone-controlled process.Structure of glycogen phosphorylase: Dimer; exists in two forms. – Active phosphorylase a – Inactive phosphorylase b Activation by phosphorylase kinase Deactivation phosphorylase phosphataseControl of phosphorylase activity: Phosphorylase kinase is activated by c-AMP protein kinase
Muscle phosphorylase In muscle, phosphorylase has 2 forms. 1. Phosphorylase a: – active form – 2 subunits, in each Ser residue at position 14 is Plated (Phosphorylase kinase does this) 2. Phosphorylase b: – inactive form – in resting muscle, all enzyme is its inactive form – structurally identical except that Ser residues are not Plated. It is active when AMP is high! It is inactive when ATP and Glc 6-P are high! So, muscle phosphorylase b is active only when the energy charge of the muscle is low. The rate of glycogen breakdown is due to the a/b which is controlled by hormones especially by epinephrine. Phosphorylase a phosphorylase b by dephosphorylation catalyzed by phosphorylase a phosphatase.
Muscle phosphorylase regulation Both phosphorylase b and phosphorylase a exist as equilibria between an active R state and a less active T state. Phosphorylase b is usually inactive because the equilibrium favors the T state. Phosphorylase a is usually active because the equilibrium favors the R state.
AMP dependency of phosphorylase b Phosphorylase a is AMP-independent Phosphorylase b is AMP-dependent. – The stimulation of phosphorylase b by AMP can be prevented by high ATP concentrations. AMP binds its allosteric site and stabilizes the conformation of phosphyrylase b in the R state. ATP acts as a negative allosteric modulator by competing with AMP and so favors the T state.Intensive exercise AMP/ATP goes up, Phosphorylase b (active)In resting muscle AMP/ATP goes down, Phosphorylase b (inactive) Exercise will also result in hormone release (epinephrine) that generates the phosphorylated a form of the enzyme.
Liver phosphorylase Liver phosphorylase and muscle phosphorylase are 90% identical in amino acid sequence. Liver phosphorylase a, but not b, has the most responsive T-to-R transition. The binding of Glc shifts the allosteric equilibrium of the a form from the R to the T state, deactivating the enzyme. Why would Glc function as a negative regulator of liver phosphorylase a? – When there is plenty of Glc, no need to breakdown liver glycogen!
Liver Glycogen phosphorylase is regulated by hormones and blood glucose. Liver glycogen has a different role in our system. – When blood glucose is low (lower than 4-5 mM) Glycogen Glc-1-P Glc-6-P Glc – So, when blood glucose is low, glucose is released into the blood stream and carried to the needy tissues. Glycogen phosphorylase of liver is under hormonal control. – Glucagon is the hormone. – When glucose is low, glucagon is released. – Liver phosphorylase is allosterically regulated by Glc not AMP. • When Glc is high in the blood, it enters hepatocytes and binds regulatory sites of the enzyme, causing conformational changes (favoring the T state). • Therefore, glycogen phosphorylase is a glucose sensor. • When Glc is high, it stops its own FORMATION.
Phosphorylase kinase is activated by phosphorylation and calcium ionsThe phosphorylase enzyme. Has a fully active form and an inactive form Has a mass of 1200 kd Consist of 4 subunits ( ) – The subunit is the source of catalytic activity. – The other subunits are regulatory subunits. Is under dual control 1. Regulated by phosphorylation – The subunit is phosphorylated by cAMP dependent PKA). 2. Partly activated by calcium levels of the order of 1 mM. – The subunit is calmodulin, a calsium sensor that stimulates many enzymes. Phosphorylase kinase has the highest activity only after both the phosphorylation of the subunit and the activation of the subunit by Ca binding.
Epi and Glucagon signal the need for glycogenbreakdown PKA activates phosphorylase kinase Glycogen phosphorylase activated Glc 1-P is made What activates PKA? – HORMONES • Glucagon • Epinephrine Signal transduction – Epi – GTP-bound G proteins – Increased cAMP – PKA increases cAMP amplifies the effects of hormones
What shuts off glycogen breakdown? This signal transduction pathway is shut down by the same pathway. How? • GTP is deactivated by its inherent GTPase activity • cAMP is converted to AMP (not a second messenger) by phosphodiesterase enzyme.
Steps in Glycogen SynthesisA. UDPG synthesis: G-6-P G-1-P G-1-P + UTP UDPG + PPiB. A primer is required for glycogen synthesis: 1. A fragment of glycogen can serve as a primer in cells whose glycogen stores are not totally depleted. 2. If a glycogen fragment is not present, glycogenin, a glycosyltransferase, serves as the primer.
Steps in Glycogen Synthesis continuedC. Elongation of glycogen chains – Glucose is transferred from UDPG to the non-reducing end of the growing chain. • New glycosidic bond between C-1 of the activated sugar and C-4 of the accepting glucosyl residue • Enzyme: glycogen synthase – If no other enzyme acts on the chain, the resulting structure is a linear molecule of glucosyl residues attached by 1-4. • Such a compound, called amylose, is found in fruits. – The UDP released when the new bond is made can be convert back. UDP + ATP UTP + ADP
Steps in Glycogen Synthesis continuedD. Creating branches in glycogen: – Amylose unbranched Glygogen branches (~8) The branches are made through the “branching enzyme”, glucosyl 4:6 transferase (amylo 1, 4 - 1,6 transglycosylase – This enzyme transfers 5 to 8 glucosyl residues from the non- reducing end to another residue by an 1,6 link. – Further elongation – Branches have two important functions a) increases the solubility of the glycogen molecule. b) The number of non-reducing ends to which new glucosyl residues can be added and thereby greatly accelerating the rate at which glycongen synthesis and degradation can occur.
Summary of the synthesis UDP-Glucose synthesis UDP-glucose phosphorylase A primer is required for glycogen synthesis (glycogenin or a fragment of glycogen) Glc units are added to the either the existing glycogen chains or glycogenin (enzyme glycogen synthase). • C-4 is the non-reducing end of glycogen chain. New glucose molecules are always added to this non-reducing terminus. Elongation of glucose chains Creating branches in glycogen (enzyme transferase) Branches have 2 functions: 1. Increase the solubility of the glycogen molecule 2. Increase the rate of glycogen synthesis
How Is Glycogen Synthesis Regulated? Glucagon and Epi promote glycogenolysis, at the same time they inhibit glycogen synthesis. – Both effects are mediated by cAMP and cAMP dependent protein kinase. – Regulated enzyme: glycogen synthase • a form: active (not phosphorylated) • b form: inactive (phosphorylated) • PKA and other kinases phosphorylate the enzyme. – Protein kinase (Ser – phosphorylated) Steps after the binding of the hormones: – Epi liver cell recep. – Adenylate cyclase activity – cAMP – cAMP Pkinase which phosphorylates and inactivates glycogen synthase
Coordinate Control of Glycogen Breakdown and Synthesis by cAMP CascadesGlycogen degradation Glycogen synthesis Inactive forms are shown in red, active forms are shown in green.
Glycogen degradationInactive forms are shown in red,active forms are shown in green.
Glycogen synthesisInactive forms are shown in red,active forms are shown in green.
Breakdown and synthesis are reciprocallyregulated Hormone -triggered cAMP cascade acting through PKA Glycogen breakdown and synthesis are reciprocally regulated. • Phosphorylase kinase also inactivates glycogen synthase. PP1(protein phosphatase 1) reverses the regulatory effects of glycogen metabolism. – PKA action is reversed by phosphatases – PP1 inactivates phosphorylase kinase and phosphorylase a by dephosphorylating these enzymes. – PP1 also removes P groups from the glycogen synthase b to the glycogen synthase a form (more active)
PP1 has 3 components: – P1 – Rgl – I How is phosphatase activity of PP1 regulated? – Rgl phosphorylation by PKA prevents its binding to PP1, therefore activation of cAMP cascade leads to the inactivation of PP1 because it cannot bind to its substrate. – Phosphorylation of inhibitor 1 by protein kinase A blocks catalysis by PP1. Thus, Epi increases glycogen breakdown by making phosphorylase a and decreases glycogen synthesis by making inactive phosphatase.
Insulin stimulates glycogen synthesisby activating protein phosphatase 1 When blood glucose is high, insulin is stimulated. Activated insulin-sensitive protein kinase makes activated protein phosphataseThe consequent dephosphorylation of glycogensynthase, phosphorylase kinase, and phosphorylasepromotes glycogen synthesis and blocks itsdegradation!
Phosphorylation of the enzymes is regulatedby hormones Phosphorylated groups can be removed by phosphatases; therefore, the action of phosphatases always opposes kinases. – If kinases activity is greater than activity of phosphatase, the enzyme is in the phosphorylated mode. Insulin, Glucagon, and Epi are three important hormones which affect glycogen metabolism!
Glycogen metabolism in the liverregulates the blood-glucose level After a carbohydrate-rich meal blood glucose increases. Insulin is the primary signal for glycogen synthesis. Blood glucose level 80-120 mg/dL (4.4-6.7 mM) The liver senses the concentration of blood glucose and either release or takes up glucose. Glucose infusion changes the enzymes involved in glycogen metabolism
Glucose regulation of glycogen metabolism Phosphorylase a is the glucose sensor in liver cells Glucose is high Binding of Glc converts R T PP1 is released Inactivation of glycogen breakdown and the activation of glycogen synthesis take place..
Summary of the Regulation ofGlycogen Synthesis and Degradation Synthesis and degradation are regulated by the same hormonal signals! An increase in insulin stimulates glycogen synthesis An increase in glucagon or Epi stimulates glycogen degradation cAMP production increases in response to the release ofEpi and glucogon cAMP production decreases in the presence of insulin! Key enzymes are phophorylated by a family of kinases, some of which are cAMP dependent. Phosphorylation of an enzyme causes 3D change that affects the active site. It may either increase or decrease its activity depending on the type of enzyme.
Glycogen storage diseases Glycogen metabolism is a finely controlled system. – It is not surprising that genetically determined enzyme deficiencies result in disease state. – Genetic diseases are in fact valuable research tools for us. There are 8 glycogen storage diseases but we will only cover Type I and Type V – Type I • Von Gierke Disease • Glc 6-phosphatase is missing. – Type V • McArdle Disease • Phosphorylase is missing.