Weill Cornell Medicine-Qatar Principles of Biochemistry Lecture 18 PDF

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJ...

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJIMI DO NOT have a financial interest in commercial products or services. Lecture 18 Glycogen metabolism Glycogen breakdown and synthesis Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 15: 556-565 STORAGE OF GLUCOSE AS GLYCOGEN The major fate of glucose in animal tissues is the breakdown in the glycolysis pathway, followed by oxidation in the citric acid cycle. Excess Glucose is converted in part into glycogen, which is an energy source stored mainly in the liver and muscle. Conversion of excess glucose in a polymeric storage form (glycogen in vertebrates and starch in plants). In vertebrates, by covalent modification, allosterically and hormonally (by insulin and glucagon) Glycogen appears as electron-dense particles, often in aggregates or rosettes (here in liver hepatocytes). GLYCOGEN METABOLISM q Excess glucose in the diet is converted mainly into two products, glycogen or fats. q Glycogen is stored largely in the liver and in muscle in large cytosolic granules. The basic glycogen particles, called β-particles, consist of up to 55000 glucose residues with in the order of 2000 non-reducing ends. 20 to 40 of these particles cluster together in the form of α-rosettes, that can be seen under a microscope in tissues from well fed animals. q Glycogen in the liver serves a a reservoir of glucose for other tissues when dietary glucose is not available. This is especially important for neurons of the brain and red blood cell that can not use fatty acids as fuel. Liver glycogen can be depleted within 12 to 24 hours. q Glycogen in muscle is there to provide a quick source of energy and the glycogen store in vigorously contracting muscle can be depleted within about an hour. Glycogen granules are complex aggregates of glycogen itself, the enzymes that synthesize and break down these molecules and the machinery that regulates these enzymes. GLYCOGEN STRUCTURE Glucose and other sugars are reducing Oxidation of the anomeric carbon C1 (and probably the neighboring carbon) of glucose and other sugars under agents capable of reducing Cu2+. alkaline conditions by Cu2+ is the basis for the Fehling's reaction. The cuprous ion (Cu+) produced forms cuprous oxide precipitate colored red. In the hemiacetal (ring) form, C-1 of glucose cannot be oxidized by Cu2+. However, in the open-chain form, which is in equilibrium with the ring form, C1 can, and eventually the oxidation reaction goes to completion. The reaction with Cu2+ is complex, yielding a mixture of products of carboxylic acids Starch: amylose+amylopectin C1 of glucose Amylose: A linear polymer of D-glucose residues in (α1→4) linkage. A single chain can contain several thousand glucose residues. Amylopectin has stretches of similarly linked residues between branch points (it is a branched amylose). Glycogen (α1→4) and (α1→6) linkages has the same basic structure as amylopectin but has more branching than amylopectin. Glycogen (highly branched) A cluster of amylose and amylopectin like that believed to occur in starch granules. Strands of amylopectin (black) form double-helical structures with each other or with amylose strands (blue). Glucose residues at the nonreducing ends of the outer branches are removed enzymatically during the mobilization of starch for energy production. Glycogen has a similar structure but is more highly branched and more compact. STRUCTURE AND FUNCTION OF SOME POLYSACCHARIDES 1/ Glycogen breakdown GLUCOSE RESIDUES ARE REMOVED FROM GLYCOGEN BY GLYCOGEN PHOSPHORYLASE TO GIVE GLUCOSE-1-P Removal of a glucose residue from the non-reducing end of a glycogen chain by glycogen phosphorylase to give glucose-1-P Glycogen à glucose 1-P (glycogen phosphorylase) Breakdown of glycogen in muscle and liver: Pyridoxal Phosphate cofactor unusual role as general acid catalyst glycogen phosphorylase cleaves the (α1→4) glycosidic link between two glucose residues at one of the non-reducing ends. This reaction is unusual in that it uses Pi rather than water in the process. As a result of phosphorolysis, rather than hydrolysis, some of the energy of the glycolysis bond is preserved in the resulting phosphate ester, glucose 1-phosphate Product of phosphorylase reaction is glucose 1P, not glucose This process is repetitive; the enzyme removes successive glucose residues until it reaches the fourth glucose unit from a branch DEBRANCHING ENZYME DEALS WITH BRANCH POINTS IN GLYCOGEN Glycogen breakdown near an (α1→6) branch point: Glycogen phosphorylase acts repetitively until it reaches a point four glucose residues away from an (α1→6) branch. At this point, the enzyme can go no further. A second enzyme is required, the debranching enzyme, that uses its transferase activity to transfers three linked residues of glucose behind a branch point to the non-reducing end of another chain. Once this has happened, the (α1→6) glucosidase activity of the debranching enzyme hydrolyzes the (α1→6) link, releasing glucose, and the process of degradation can continue with glycogen phosphorylase à glucose 1-P. GLUCOSE-1-PHOSPHATE MUST BE ISOMERIZED TO GLUCOSE-6-PHOSPHATE FOR METABOLISM glucose 1-P à glucose 6-P (phosphoglucomutase) The Reaction is catalyzed by phosphoglucomutase. The reaction begins with the enzyme phosphorylated on a Ser residue. In step 1, the enzyme donates its phosphoryl group (blue) to glucose 1-phosphate, producing glucose 1,6bisphosphate. In step 2, the phosphoryl group at C-1 of glucose 1,6-bisphosphate (red) is transferred back to the enzyme, reforming the phosphoenzyme and producing glucose 6-phosphate, that can enter the glycolysis pathway in muscle. GLUCOSE-6-PHOSPHATE IS DEPHOSPHORYLATED IN THE LIVER FOR TRANSPORT OUT OF THE LIVER Glucose 6-phosphate is hydrolyzed by glucose 6-phosphatase of the ER to glucose that enters the bloodstream The catalytic site of glucose 6phosphatase faces the lumen of the ER. A glucose 6-phosphate (G6P) transporter (T1) carries the substrate from the cytosol to the lumen, and the products glucose and Pi pass to the cytosol on specific transporters (T2 and T3). Glucose leaves the cell via the GLUT2 transporter in the plasma membrane. § The liver has a different function (compared to muscle) which is to supply other tissues with glucose. Glucose 6-P is transported into the lumen of the ER where the enzyme glucose 6-phosphatase hydrolyzes it to glucose and phosphate which are then transported separately back into the cytosol. From there glucose is released into the bloodstream via the GLUT2 transporter. § Muscle and adipose tissue lack glucose 6 phosphatase (so no gluconeogenesis) and can therefore not contribute glucose to the blood. 2/ Glycogen synthesis FOR GLYCOGEN SYNTHESIS, THE SUGAR NUCLEOTIDE UDP-GLUCOSE DONATES GLUCOSE Initiation of glycogen synthesis (starts with glucose 6-P) glucose 6-P ßàglucose 1-P (phosphoglucomutase) glucose 1-P + UTP à UDP-glucose + PPi (UDP-glucose phosphorylase) The polymerization of hexoses to disaccharides, glycogen, starch, cellulose or other complex extracellular polysaccharides involves sugar nucleotides as intermediates. One example is uridine diphosphate glucose (UDP-glucose) in the synthesis of glycogen. Formation of a sugar nucleotide: A condensation reaction occurs between a nucleoside triphosphate (NTP) and a sugar phosphate. The negatively charged oxygen on the sugar phosphate serves as a nucleophile, attacking the phosphate of the nucleoside triphosphate and displacing pyrophosphate. The reaction is pulled in the forward direction by the hydrolysis of PPi by inorganic pyrophosphatase. GLYCOGEN IS SYNTHESIZED BY GLYCOGEN SYNTHASE UDP-glucose is the donor of glucose residues to a non-reducing end of a glycogen branch (UDP glucose is the substrate for glycogen synthase) Glycogen Chain Elongation UDP-glucose is the immediate donor of glucose residues in a reaction catalyzed by the enzyme glycogen synthase. The enzyme transfers the glucose residue of UDP-glucose to the non-reducing end of a glycogen branch to make a new (α1→4) linkage. The overall equilibrium from glucose 6-phosphate to a lengthened glycogen favors synthesis. SYNTHESIS OF BRANCHES IN GLYCOGEN BY THE BRANCHING ENZYME glycogen synthase cannot make the a1à 6 bonds found at the branch point of glycogen: These are formed by the glycogen branching enzyme Glycogen synthase can only elongate an existing chain but cannot introduce (α1→6) bonds at the branch points of glycogen. Therefore, a second enzyme is required that removes a fragment of 6 or 7 residues of glucose from a chain, at least eleven units long, and transfers this to the C-6 hydroxyl group of a glucose residue more interior to the molecules. This provides the basis for extending the chain once more with glycogen synthase. Branch synthesis in glycogen. The glycogen-branching enzyme (also called amylo (1→4) to (1→6) transglycosylase, or glycosyl-(4→6)-transferase) forms a new branch point during glycogen synthesis. GLYCOGENIN PRIMES THE INITIAL SUGAR RESIDUES IN GLYCOGEN glycogen synthase cannot initiate a new glycogen chain de novo, it requires a primer. Glycogenin is both the primer and the enzyme that catalyzes the formation of a new chain Glycogen synthase cannot initiate glycogen chains de novo. This enzyme requires a preformed (α1→4) polyglucose chain or a branch at least 8 glucose residues long. Glycogenin structure A protein, called glycogenin, is used both as the primer on which new chains are assembled and the enzyme that catalyzes their assembly. Muscle glycogenin (Mr 37,000) forms dimers in solution. Humans have a second isoform in liver, glycogenin-2. The substrate, UDP-glucose (shown as a red ball-and-stick structure), is bound near the amino terminus and is some distance from the Tyr194 residues (turquoise). Each UDP-glucose is bound through its phosphates to a Mn2+ ion (green) that is essential to catalysis. The glycosidic bond in the product has the same configuration about the C-1 of glucose as the substrate UDP-glucose, suggesting that the transfer of glucose from UDP to Tyr194 occurs in two steps. The first step is probably a nucleophilic attack by Asp162 (dark blue), forming a temporary intermediate with inverted configuration. A second nucleophilic attack by Tyr194 then restores the starting configuration GLYCOGENIN CATALYZES TWO DISTINCT REACTIONS Glycogenin catalyzes two distinct reactions: 1/ Initial attack by the hydroxyl group of Tyr194 on C-1 of the glucosyl moiety of UDP-glucose results in a glucosylated Tyr residue. 2/ The C-1 of another UDP-glucose molecule is now attacked by the C-4 hydroxyl group of the terminal glucose, and this sequence repeats to form a nascent glycogen molecule of eight glucose residues attached by (α1→4) glycosidic linkages. At this point glycogen synthase can take over to add UDP glucose and make glycogen. GLYCOGENIN AND THE STRUCTURE OF GLYCOGEN PARTICLE Starting at a central glycogenin homodimer, molecule, glycogen chains (12 to 14 residues) extend in tiers. - Inner chains (B-chains) have two α1→6 branches each - A-chains in the outer tier are unbranched There are (in theory) 12 tiers in a mature glycogen particle (only 5 are shown here), consisting of about 55000 glucose in a molecule of about 21 nm diameter and Mr ≈1x107. JC08: GLYCOGEN STORAGE DISEASES OF HUMANS Remember to prepare for next lecture: Lehninger’s Biochemistry (8th ed), §chapter 13: p. 496-503