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PRINCIPLES OF METABOLIC REGULATION: GLUCOSE AND GLYCOGEN Lehninger Ch. 15 BIO 322 Recitation 2 / Spring 2013 •Glycogen stored - %10 weight of the liver •0.4 M in a hepatocyte if dissolved as glucose – impaired osmotic properties (As glycogen 0.01 μM) •Liver glycogen can be depleted 12-24 hrs •Muscle glycogen – less than a hour Catabolic: Glycogen – G6P (glycogenolysis) G6P – pyruvate (glycolysis) Anabolic: Glucose – Glycogen (glycogenesis) Pyruvate – glucose (gluconeogenesis) Glycogen phosphorylase (α1-4 glycosidic linkage at a non-reducing end) Different from hydrolysis by amylase at the intestine. Phosphorolysis reaction Some of the energy of the glycosidic bond is preserved in the product, G1P Pyridoxial phosphate (Vitamin B6) – cofactor essential for glycogen phosphorolyase, promote attack by Pi on glycosidic bond. Repetitive action on non-reducing ends of the glycogen branches until four glucose residue away from branch point (α1-6). Debranching enzyme ( oligo (α1-6) to (α1-4) glucatransferase) 2 Debranching enzyme ( oligo (α1-6) to (α1-4) glucatransferase) Tranferase + glucosidase activity After all, Glycogen phosphorylase activity can continue on. 3 Phosphoglucomutase G1P to G6P (Reversible rxn) Phosphorylated enzyme transfers P to C6 (G1,6P) P on C1 back to enzyme, reformed phosphoezyme. G6P to glycolysis (in muscle) 4 The active site of Glucose 6Phosphatase separates from glycolysis Glycogen breakdown in liver is to release glucose to blood when blood glucose is low. 5 Glucose 6-phosphatase (only in liver and kidneys, not in muscle and in adipose), integral membrane protein in ER, active site at lumenal side. Cytosolic G6P into ER lumen by T1 Glucose out from ER lumen by T2 Phosphate out from ER lumen by T3 Glucose leave the cell via GLUT2 into blood stream. G6P phosphatase in ER maintains blood glucose levels, rather than just directing G6P directly to glycolysis. Sugar nucleotides are suitable for biosynthetic rxns. (Substrates for polymerization of monosac to disacch., glycogen, starch, cellulose, vitamin C) 1) Their formation is metabolically irreversible, contributing to the irreversibility of the biosynthetic pathways in which they are intermediates. 2) Nucleotide moiety can significantly contribute to catayltic activity of the enzymes • Groups can undergo noncovalent interactions with enzymes. 3) Nucleotidyl group (AMP, UMP) is excellent leaving group, activates the sugar carbon to which it is attached for nucleophilic attack. 4) Cells can differentiate hexoses with different nucleotidyl groups from hexose phosphates. 6 Glycogen Synthesis 1. Glycogen synthesis starts with Glucose-6-Phosphate by hexokinase I and II in muscle and IV in liver. Cori cycle – erythrocytes take up glucose, convert it to lactate, this lactate is then taken up by liver and converted to G6P by gluconeogenesis. (Fate of some ingested glucose) 7 2. Phosphoglucomutase – G6P to G1P 3. UDP-glucose pyrophophorylase G1P + UTP UDP-glucose +PPi 4. Glycogen synthase – Transfer of glucose residue to non-reducing ends. Phosphoglucomutase – G6P to G1P UDP-glucose pyrophophorylase (names for the reverse rxn) G1P to + UTP UDP-glucose +PPi 8 UDP-glucose formation has positive free energy PPi hydrolysis (by pyrophosphatase) is strongly exergonic to keep PPi concentration low in cell. Glycogen Synthase cannot make α16 bonds at the branching points Amylo (14) to (16) transglycosylase or glycosyl (46) transferase At least 11 residues, take 6 or 7 residues to make a branch point. The biological significance is to make glycogen more soluble and to increase the # of non-reducing ends. This increases the available sites for glycogen phosphorylase and glycogen synthase, both which act at non-reducing ends. 9 •Glycogen synthase cannot initiate glycogen synthesis de novo. •A primer is required, usually a preformed (α14) polyglucose chain or 8 glucose residue containing branch. •Protein called glycogenin. •Glucosyltransferase activity (intrinsic) – glucose of UDP-glucose to Tyr194 of glycogenin. •Chain extending activity of glycogenin – Add 7 more glucose residues. 10 •Glycogen synthase continues to add glucose, glycogenin remains buried. •AMP concentration is much more sensitive indicator of cell`s energetic state than ATP. •In cells, ATP – 5 to 10 mM, AMP (<0.1mM) •When muscle consumes ATP, AMP is produced in 2 steps. •First, hydrolysis of ATP produces ADP •Then, adenylate kinase produces AMP. •If ATP concentration drops by 10%, producing ADP and AMP in the same amounts, the relative change in AMP is much greater. Hence, regulation is easier by looking at changes in AMP concentration. •AMPK (AMP dependent protein kinase) •Active AMPK increases glucose transport, activates glycolysis and FA oxidation, supresses energy requiring processes such as FA, cholestrol and protein synthesis. 11 12 Gluconeogenesis – primarily in liver. Hexokinase, PFK-1 and Pyruvate kinase All the rxns. Carried out by these enzymes are highly exergonic. Glucose 6 phosphatase Fructose 1,6- biphosphatase Pyruvate carboxylase and PEP carboxykinase Futile cycle vs. Substrate Cycle 13 Hexokinase (4 isozymes) Hexokinase II, predominant in myocytes – Km 0.1 mM (blood glucose 4-5 mM), high enough to saturate hexokinase II. Muscle hexokinases I and II, allosterically inhibited by their product G6P. (Allosteric Regulation example) Muscle consumes glucose Liver maintains blood glucose homeostatis 14 Hexokinase IV in liver (glucokinase) differ from I,II and III in three respects: 1) Km is 10 mM which is higher than blood glucose. (Active at higher concentrations of glucose, a protective role ?) 2) Sequestration in nucleus when F6P concentration is high. When glucose is high back to cytosol. (Sequestration, assosication with regulatory protein example) 3) Not inhibited by G6P, continues to operate. 15 PFK-1 – irreversible rxn that commits glucose to glycolysis. ATP inhibits PFK-1 by binding to an allosteric site, lowers affinity to F6P. Citrate, a key intermediate of pyruvate oxidation, FA and AA, increases the allosteric inhibiton by ATP. 16 PFK-1 is activated by F2,6BP (Most significant) Signs of abundant energy supplys (ATP, acetyl-coA, long chain FA), allosterically inhibit all isozymes of pyruvate kinase. 17 L form in liver – When blood glucose is low, thus causing glucagon release, cAMPdependent protein kinase (PKA) phosphorylates L isozyme of pyruvate kinase and inactivates it. Slows down the glucose usage in liver and spares it for export to brain and other organs. In muscle, in response to epinephrine, increased cAMP causes glycogen breakdown, glycolysis and provides fuel to the cell for fight or flight response. Gluconeogenesis Regulation 18 FA breakdown – product acetyl-CoA Acetyl-CoA – positive pyruvate carboxylase Negative pyruvate dehyrogenase complex Rapid, instantly irreversible allosteric mechnanisms in Miliseconds vs. Seconds and minutes via hormones Not just a futile cycle, but links glycolysis to gluconeogenesis. 19 Glycogen Breakdown Resting | Contracting Glycogen phosphorylase is regulated at allosteric and hormonal levels. Glucagon and Epi – cAMP high High cAMP conc PKA phopshorylase b kinase Ser residues of glycogen phosphorylase In contracting muscle, Ca binds to calmodulin residue of phophorylase b kinase. High AMP due to this muscle contraction further activates glycogen phosphorylase. 20 21 Glycogen Synthase GSK3 cannot phosphorylate glycogen synthase until another protein kinase, casein kinase II has first phosphorylated the glycogen synthase on a nearby residue, an event called priming. Glucose 6 phosphate is the allosteric activator. (GS acts G6P sensor) 22 23 Central Role of PP1 24 25 26