161718192021Role of citric acid cycle in anabolismAnaplerotic rxns (in red) replenish depleted cycle intermediates22Feeder pathways for glycolysis hexosecatabolismGal(β1->4) Glclivermusclestorage polysaccharidesGlc(α1<->α4) GlcFr(β2<->α1) GlcGlc(α1->4) Glc23Glycogen granules associate with the smooth endoplasmic reticulum in the cytoplasmSmooth ERRough ERMitoGlycogen GranulesGlycogen makes up 7-10% wet weight of liver and 1-2% wet weight of a muscle cell. Equivalent concentration of glucose is 0.4 M but because it is a polymer and insoluble, it actually contributes to a concentration of 0.1 microM in the cell. Glycogen is stored in large cytoplasmic granules in the cell, associated with the smooth endoplasmic reticulum. The glycogen particle has a higher order structure. The Beta-particle is 21 nm in diameter and consists of 55,000 glucose residues with about 2,000 non-reducing ends. 20 to 40 particules cluster together to form an alpha-rosette, which is seen in the electron micrograph of a well-fed animal hepatocyte (liver cell). After a 24 hour fast, essentially no glycogen particles will be detected. The glycogen is organized with the enzymes that catalyze synthesis and degradation as a mini-factory within the cells. The enzymes that work on glycogen are NOT floating freely throughout the cell!24Glycogen structureStorage polysaccharideα1->4 polymer of glucose with α1->6 branchesmore extensively branched than starch and more compactstored in liver, muscle ave MW 7 million25Glycogen BreakdownGlucose mobilized from nonreducing endphosphorolysis reaction G-1-P (P maintains energy)Glu-6-PGlycogen phosphorylase catalyzes phosphorolytic cleavage of the alpha (1Æ4) glycosidic linkages of glycogen, releasing glucose-1-P as a reaction productGlycogen (n residues) +Pi Æ glycogen (n-1) + glucose-1-PPhophorolysis may be compared to hydrolysis:Hydrolysis R-O-R’ + HOH Æ R-OH and R’-OHPhosphorolysis: R-O-R’ + HO-PO3 2- Æ R-OH + R’-O-PO3 2-Glycogen Phosphorylase has Pyridoxal Phosphate (PLP), a derivative of Vit B6 as a prosthetic group.PLP is held at the active site of the phosphorylase enzyme by a Schiff base linkage, formed by reaction of the aldehyde of PLP with the epsilon amino group of a lysine residueIn contrast to the role of this cofactor in other enzymes, the phosphate of PLP is involved in acid/base catalysis by Phosphorylase. The Pi substrate binds between the phosphate of PLP and the glycosidic O linking the terminal glucose residue of the glycogen. After the phosphate substrate donates H+ during cleavage of the glycosidic bond, it receives H+ from the phosphate moiety of PLP.PLP then takes back the H+ as the phosphate O attacks C1 of the cleaved glucose to yield glucose-1-phosphate26A glucose analog, N-acetylglucosamine (GlcNAc), is adjacent to pyridoxal phosphate at the active site in the crystal structure shown. Glycogen Phosphorylase:A homodimericenzyme, subject to allosteric control. Crystal structure: PLP PLP GlcNAc GlcNAc inhibitor Human Liver Glycogen Phosphorylase PDB 1EM627Question: Why would an inhibitor of Glycogen Phosphorylase be a suitable treatment for diabetes?A class of drugs developed for treating the hyperglycemia of diabetes (chloroindole-carboxamides), inhibit liver Phosphorylase allosterically.These inhibitors bind at the dimer interface, stabilizing the inactive conformation. PLP PLP GlcNAc GlcNAc inhibitor Human Liver Glycogen Phosphorylase PDB 1EM6 A glycogen storage site on the surface of the Phosphorylase enzyme binds the glycogen particle. Given the distance between the storage and active sites, Phosphorylase can \ cleave alpha (1Æ4) linkages only to within 4 residues of an alpha (1Æ6) branch point. This is called the limit branch.28cleaves until 4 glucoses from a branch point debranching enzyme -2 functions1. transferase activity2. α1->6 glucosidasePhosphorylase can act again until 4 glucoses from a branch pointhydrolysis rxnDebranching enzyme has 2 independent active sites consisting of residues indifferent segments of a single polypeptide chain.The transferase of the debranching enzyme transfers 3 glucose residues from a 4-residue limit branch to the end of another branch, diminishing the limit branch to a single glucose residue.The alpha (1Æ6) glucosidase moiety of the debranching enzyme then catalyzes hydrolysis of the alpha (1Æ6) linkage, yielding free glucose. This is a minor fraction of the glucose released from glycogen.The major product then of glycogen breakdown is glucose-1-phosphate, from phosphorylase activitiy.29Glc-1-P to Glc-6-P and then glycolysis, mxn of phosphoglucomutase:reversible reactionmxn similar to phosphoglyceromutase(except E-His)G-6-P in liver to glycolysis or pentose P pathway or to other tissues. Serine is phosphorylated and de-phosphorylatedin the catalytic cyclePhosphoglucomutase catalyzes the reversible reaction:Glucose 1-P Å> glucose -6-PA serine OH at the active site donates and accepts Pi. The bisphosphate is not rreleased. Phosphoglycerate mutase has a similar mechanism, but instead uses His for Pi transferThe product glucose-6-p may enter glycolysis or (in liver) be dephosphorylated for release to the blood.30 Glycogen Glucose Hexokinase or Glucokinase Glucose-6-Pase Glucose-1-P Glucose-6-P Glucose + Pi Glycolysis Pathway Pyruvate Glucose metabolism in liver. To bloodRole in maintainingBlood [glucose] steady(from blood, i.e. after eating)phosphoglucomutaseThe reversible phosphoglucomutase may also convert glucose-6-P Æglucose -1-P, precursor for glycogen synthesis as well as glycogen breakdown product. Liver glucose-6-P catalyzes the following , essential to the liver’s role in maintaining blood glucose:Glucose-6-P + H2O Æ glucose + PiMost other tissues lack this