Glycogen Metabolism Dr. Mohammad Saadeh

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1 Glycogen Metabolism Presented by Dr. Mohammad Saadeh The requirements for the Pharmaceutical Biochemistry II Philadelphia University Faculty of pharmacy

2 I. overview Glucose is energy source for Brain. RBCs (no mitochondria). Exercising muscle, because the glucose is the substrate for anaerobic glycolysis. Blood glucose coming from three sources: Diet (glucose, monosaccharides, disaccharides, polysaccharide such as starch. Disaccharides. Degradation of glycogen. Glycogen stores in: Liver and kidney. Skeletal muscle.

3 Structure and Function of Glycogen: A. Amounts of liver and muscle glycogen 1 2% of weight of resting muscle, and 10% of weight of a well-fed adult liver. B. Structure of Glycogen Glycogen is a branched polysaccharide made from α-d-glucose. bonded by α(1 4) linkage. After 8-10 glucosyl residues, there is a branch α(1 6) linkage. C. Function of glycogen stores Glycogen synthesis of ATP during muscle contraction. Glycogen liver used to maintain the blood glucose concentration during the early stages of a fast.

4 Glycogen synthesized from α-d-glucose. Glycogenesis occurs in the cytosol. Glycogenesis (synthesis of glycogen) Glycogenes is requires energy supplied by ATP and uridine triphosphate (UTP). A. Synthesis of UDP-glucose: building block of glycogen is UDP-glucose synthesized from glucose 1-phosphate and UTP by UDP-glucose pyrophosphorylase (Figure 11.5). B. Synthesis of a primer to initiate glycogen synthesis: glycogen fragment called glycogenin, accept of glucose residues from UDP-glucose (Figure 11.5). producing a short, α(1 4)- linked glucosyl chain and elongated by glycogen synthase. UDP-glucose: building block of glycogen

5 Glycogenesis (synthesis of glycogen) C. Elongation of glycogen chains by glycogen synthase (Figure 11.5). Elongation of a glycogen by transfer of glucose from UDP-glucose to the non-reducing end of the growing chain by glycogen synthase that is making the α(1 4) linkages. Then, D. Formation of branches in glycogen (Figure 11.5). amylo-α(1 4) α(1 6)-transglucosidase (4:6 transferase), transfers a chain of six to eight glucosyl residues from the nonreducing end of the glycogen chain by breaking an α(1 4) linkage, and attaches it with an α(1 6) linkage to another residue in the chain.

6 Figure 11.5 Glycogen synthesis.

7 Degradation of Glycogen (Glycogenolysis) The degradative pathway for stored glycogen in liver and skeletal muscle accrue by separate set of cytosolic enzymes. Steps of glycogen degradation: A. Shortening of chains: Glycogen phosphorylase (need pyridoxal phosphate as coenzyme) cleaves the α(1 4) bonds at glycosyl residue at non-reducing ends of the glycogen chains, producing glucose 1-phosphate. The resulting structure is called a limit dextrin. B. limit dextrin that is degraded by the bifunctional debranching enzyme: 1. oligo-α(1 4) α(1 4)-glucan transferase activity (or glucosyl 4:4 transferase) remove the three of four glycosyl residue attached to the branch. 2. amylo-α(1 6)-glucosidase activity removed glucose residue attached in an α(1 6) linkage. C. Glucose 1-phosphate converted to glucose 6-phosphate by phosphoglucomutase, then glucose 6-phosphate converted to glucose by glucose 6-phosphatase. D. (1-3% ) of glycogen degraded by lysosomal enzyme (α(1 4)-glucosidase (acid maltase).

8 Degradation of Glycogen (Glycogenolysis)

9 Degradation of Glycogen (Glycogenolysis)

10 Regulation of Glycogenesis and Glycogenolysis During fast (low glucose level, glucagon & epinephrine are elevated that cause stimulate Glycogenolysis (glycogen degradation). But when elevated glucose, the insulin will be increased. A. Stimulation of Glycogenolysis (glycogen degradation ); figure 11.9: 1. low level of glucose glucagon & epinephrine bonded to G protein receptor active adenylyl cyclase generate camp active protein kinase A (phosphorylation of glycogen phosphorylase kinase; active) (phosphorylation of glycogen phosphorylase; active) stimulate glycogen degradation (glycogenlysis). 2. During muscle contraction, Ca +2 is released from endoplasmic reticulum. Ca +2 bind to caldmodulin subunit of phosphorylase kinase b, activating it without phosphorylation. Phosphorylase kinase can then activate glycogen phosphorylase, causing glycogen degradation. 3. AMP activates glycogen phosphorylase b without phosphorylated. B. Inhibition of Glycogenolysis(glycogen degradation ); figure 11.9: High level of glucose Insulin elevated activate phosphodiesterase camp convert to 5ˋ-AMP AMP activated protein phosphatase-1 remove phosphate group from both glycogen phosphorylase kinase a and glycogen phosphorylase a to become inactive inhibit glycogen degradation (glycogenlysis).

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13 Regulation of Glycogenesis and Glycogenolysis A. Inhibition of Glycogenesis (glycogen synthesis); figure : 1. low level of glucose glucagon & epinephrine bonded to G protein receptor active adenylyl cyclase generate camp active protein kinase A (phosphorylation of glycogen synthase kinase a (active); forming glycogen synthase kinase b (inactive) inhibited glycogen synthesis (glycogenesis). B. Stimulation of Glycogenesis (glycogen synthesis); figure : High level of glucose Insulin elevated activate phosphodiesterase camp convert to 5ˋ-AMP AMP activated protein phosphatase-1 remove phosphate group from both glycogen synthase b (inactive) forming glycogen synthase a (active) stimulate glycogen synthesis (glycogenesis).

14 Allosteric regulation of glycogen synthesis and degradation A. Allosteric Stimulate glycogen synthesis (glycogenesis) by activated glycogen synthase Stimulate by glucose 6-P B. Allosteric Inhibition of glycogen degradation (glycogenlysis) by inhibited glycogen phosphorylase Glucose 6-P ATP Glucose C. Allosteric stimulation of glycogen degradation (glycogenlysis) by activated glycogen phosphorylase: Ca+2 AMP in muscle

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16 Pentose Phosphate Pathway and Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Presented by Dr. Mohammad Saadeh The requirements for the Pharmaceutical Biochemistry II Philadelphia University Faculty of pharmacy

17 Overview Pentose phosphate pathway (also called the hexose monophosphate shunt) that occurs in the cytosol. It oxidize G6-Phosphate to produce CO 2 and 2NADPH (reducing agent) and ribose5-phosphate required for biosynthesis of nucleotides such as (DNA, RNA, ATP, NADH, FAD, and CoA) No ATP is directly consumed or produced in the cycle. Unlike NADH, NADPH contain a phosphorylated 2ˋ-hydroxyl group on one of the ribose unit.

18 pentose phosphate pathway consists of two phases: A. Irreversible oxidative reaction Glucose 6-P +2NADP + +H 2 O Ribose5-phosphate + 2NADPH + CO 2 + 2H + B. Reversible nonoxidative reactions Ribose5-phosphate Synthesis of Ribose5-phosphate to synthesis (DNA, RNA, ATP, NADH, FAD, and CoA) OR synthesis intermediates for glycolysis

19 A. Irreversible oxidative reaction 1. Dehydrogenation of glucose 6-phosphate (rate limiting): G6PD Glucose 6-P + NADP + +H 2 O 6-phosphogluconate + NADPH Glucose 6-phosphate dehydrogenase (G6PD) catalyzes an irreversible oxidation of glucose 6-phosphate to 6-phosphogluconolactone and forming NADPH. Regulated of pentose phosphate pathway (PPP): NADPH is a competitive inhibitor for G6PD. Therefore, when NADPH/NADP + increase cause inhibit G6PD (inhibit PPP). Insulin stimulate expression of the gene for G6PD (activate PPP). 2. Formation of ribulose 5-phosphate 6-phosphogluconate undergoes oxidative decarboxylation by 6-phosphogluconate dehydrogenase to produce CO 2 (from carbon 1 of glucose) two molecule of NADPH pentose sugar phosphate (ribulose 5-phosphate). G6PD 1) Glucose 6-P + NADP + +H 2 O 6-phosphogluconate + NADPH 6-phosphogluconate dehydrogenase 2) 6-phosphogluconate + NADP + Ribose5-phosphate + NADPH + CO 2 + 2H + TOTAL = CO 2 + Ribose5-phosphate + 2NADPH

20 B. REVERSIBLE NONOXIDATIVE REACTIONS occur in cell to synthesis (ribose 5-phosphate for nucleotides, nucleic acids) and (intermediates of glycolysis such as glyceraldehyde 3-phosphate and fructose 6- phosphate). 1. Two reversible isomerization reaction Ketose to aldose conversion (Ribulose 5- phosphate convert to Ribose 5-phosphate that is included in nucleotides and nucleic acids synthesis by ribose 5-phosphate isomerase. Inversion configuration of Ribulose 5- phosphate to produce xylulose 5- phosphate by phosphopentose epimeras. nucleotides, nucleic acids ribose 5-phosphate isomerase phosphopentose epimeras

21 B. REVERSIBLE NONOXIDATIVE REACTIONS 2. Transketolase reaction I: Transketolase catalyse by coenzyme called thiamine pyrophosphate (TPP) to transfer two carbon from xylulose 5-phosphate to Ribose5-phosphate. These reaction produce glyceraldehyde 3-phosphate and seven carbons sugar. Transketolase TPP 3. Transaldose catalyzes transfer three carbon from seven carbon sugars to glyceraldehyde 3-phosphate, producing fructose 6-phosphate and erythrose 4 phosphate.

22 B. REVERSIBLE NONOXIDATIVE REACTIONS 4. When need ribose 5-phosohate more than NADPH: synthesis of ribose 5-phosphate from glyceraldehyde 3-phosphate and fructose 6-phosphate in the absence of the oxidative reactions.

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24 Uses of NADPH A. Reductive biosynthesis such as production of fatty acid and steroid hormone B. Reduction of hydrogen peroxide and reduction of glutathione. C. Cytochrome P450 monooxygenase system for synthesis of steroid hormone and drugs detoxification. D. Phagocytosis by white blood cells. E. Synthesis of nitric oxide.

25 A. Reductive biosynthesis: Uses of NADPH NADPH is high energy molecule such as NADH. NADPH are used in reactions as electron donor in reductive biosynthesis such as fatty acid and steroid hormone synthesis. What is the different between NADH and NADPH? Electrons of NADH transfer to oxygen in electron transport chain to produce ATP.

26 Uses of NADPH Hydrogen peroxide (ROS) produced during aerobic metabolism and can cause damage to DNA and proteins. These ROS implicated in pathologic processes. B. Reduction of hydrogen peroxide: 1. Enzymes that catalyze antioxidant reactions: NADPH required for the reduction of hydrogen peroxide, providing the reducing equivalents required by glutathione (GSH). GSH is used by glutathione peroxidase to reduce peroxide to water. The oxidized glutathione (G-S-S-G) is reduced by glutathione reductase, using NADPH as the source of electrons.

27 C. Cytochrome P450 monooxygenase system & NADPH synthesis steroide hormone and detoxify a drugs. Monooxygenases incorporate one atom from O 2 into a substrate (creating a hydroxyl group), with the other atom being reduced to water. R-H + O2 + NADPH + H+ R-OH + H2O + NADP+ R = Steroid or drugs NADPH provides reducing equivalents for the mitochondrial cytochrome P450 monooxygenase system, which is used in steroid hormone synthesis in steroidogenic tissue, bile acid synthesis in liver, and vitamin D activation in liver and kidney. Microsomal system: cytochrome P450 monooxygenase in (smooth ER) uses NADPH to detoxify foreign compounds (xenobiotics), such as drugs and a variety of pollutants by hydroxylated toxins to increase the solubility and excrete in urine or feces.

28 D. Phagocytosis by white blood cells: NADPH oxidase uses O 2 and NADPH electrons to produce superoxide radicals (O 2.- ), which converted to hydrogen peroxide (H 2 O 2 ) by superoxide dismutase. Myeloperoxidase catalyzes the formation of bactericidal hypochlorous acid from H 2 O 2 and chloride ion (Cl - ). The H 2 O 2 reduced to the hydroxyl radical (OH ). Note: genetic defect in NADPH oxidase causes chronic granulomatous disease.

29 E. Synthesis of nitric oxide (NO): Arginine, O2, and NADPH are substrates for cytosolic NO synthase (NOS) for synthesis NO. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), heme, and tetrahydrobiopterin are coenzymes for NO synthase (NOS) FMN, FAD, Heme tetrahydrobiopterin (NO), an important molecule that causes: 1.vasodilation by relaxing vascular smooth muscle. 2.Neurotransmitter. 3.Prevents platelet aggregation. 4.helps mediates tumoricidal macrophage bactericidal activity.

30 Glucose 6-phosphate dehydrogenase deficiency Missense mutation in G6PD lead to Glucose 6- phosphate dehydrogenase deficiency impairs ability of cell to form NADPH that is essential for maintenance GSH pool. Therefor; decrease in the cellular detoxification of free radicals and H 2 O 2 in cell (Figure 13.10). Forming Heinz bodies (denatured protein) in RBCs and removed in spleen due to Oxidation of sulfhydryl groups (Figure 13.11). G6PD deficiency lead to hemolytic anemia. Figure Figure 13.10

31 Glucose 6-phosphate dehydrogenase deficiency RBCs use pentose phosphate pathway only to generate NADPH therefore, G6PD deficiency lead to hemolytic anemia and neonatal jaundice in babies. Precipitating factors in G6PD deficiency G6PD mutations do not show clinical manifestations. However, some patients with G6PD deficiency develop hemolytic anemia if they are treated with: an oxidant drug, ingest fava beans, or contract a severe infection. 1. Oxidant drugs: cause hemolysis in G6PD deficiency. 2. Favism: the hemolytic effect of ingesting fava beans in all patients with favism have G6PD deficiency. 3. Infection: Inflammatory response to infection generate free radicals in macrophages, which can diffuse into the red blood cells and cause oxidative damage & hemolysis in G6PD deficiency.

32 Science Should be as simple as possible, but not simpler. Albert Einstein

33 References: Biochemistry. Lippincott's Illustrated Reviews. 6 th Edition by, Richard A Harvey, Denise R. Ferrier. Lippincott Williams and Wilkins, a Wolters kluwer business