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Lecture 24

Lecture 24. Quiz Mon. on Pentose Phosphate Pathway Glycogen regulation Quiz next Fri. on TCA cycle. Figure 23-25 The pentose phosphate pathway. Page 863. Glycogen biosynthesis. Most important storage form of sugar

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Lecture 24

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  1. Lecture 24 • Quiz Mon. on Pentose Phosphate Pathway • Glycogen regulation • Quiz next Fri. on TCA cycle

  2. Figure 23-25 The pentose phosphate pathway. Page 863

  3. Glycogen biosynthesis Most important storage form of sugar Glycogen - highly branched (1 per 10) polymer of glucose with (1,4) backbone and (1,6) branch points. More branched than starch so more free ends. Average molecular weight -several million in liver, muscle. 1/3 in liver (more concentrated but less overall mass (5-8%)), 2/3 in muscle (1%). Not found in brain - brain requires free glucose (120 g/ day) supplied in diet or from breakdown of glycogen in the liver. Glucose levels regulated by several key hormones - insulin, glucagon.

  4. Figure 18-1a Structure of glycogen. (a) Molecular formula. Page 627

  5. Figure 18-1b Structure of glycogen. (b) Schematic diagram illustrating its branched structure. Page 627

  6. Glycogen is an efficient storage form UDP-glucose G-6-P Glycogen + UDP + Pi G-1-P + UTP UDP + ATP UTP + ADP Net: 1 ATP required 90% 1,4 residues Glycogen + Pi G-1-P G-6-P Glycogen 10% 1,6 residues glucose 1.1 ATP/38 ATP so, about a 3% loss, therefore it is about 97% efficient for storage of glucose

  7. Glycogen biosynthesis 3 enzymes catalyze the steps involved in glycogen synthesis: UDP-glucose pyrophosphorylaseGlycogen synthase Glycogen branching enzyme

  8. Glycogen biosynthesis MgATP MgADP HK Glucose [G-1,6-P2] G-6-P G-1-P phosphoglucomutase F-6-P PGI The hydrolysis of pyrophosphate to inorganic phosphate is highly exergonic and is catalyzed by inorganic pyrophosphatase PPase 2Pi UTP PPi G-1-P UDP-Glucose Pyrophosphorylase

  9. Figure 18-6 Reaction catalyzed by UDP–glucose pyrophosphorylase. Page 633

  10. UDP-Glucose pyrophosphorylase Coupling the highly exergonic cleavage of a nucleoside triphosphate to form PPi is a common biosynthetic strategy. The free energy of the hydrolysis of PPi with the NTP hydrolysis drives the reaction forward.

  11. Glycogen synthase In this step, the glucosyl unit of UDP-glucose (UDPG) is transferred to the C4-OH group of one of glycogen’s nonreducing ends to form an (1,4) glycosidic bond. Involves an oxonium ion intermediate (half-chair intermediate) Each molecule of G1P added to glycogen regenerated needs one molecule of UTP hydrolyzed to UDP and Pi. UTP is replenished by nucleoside diphosphate kinase UDP + ATP UTP + ADP

  12. Figure 18-7 Reaction catalyzed by glycogen synthase. O Page 633

  13. Glycogen synthase All carbohydrate biosynthesis occurs via UDP-sugars Can only extend an already (1,4) linked glucan change. First step is mediated by glycogenin, where glucose is attached to Tyr 194OH group. The protein dissociates after glycogen reaches a minimum size.

  14. Glycogen branching Catalyzed by amylo (1,41,6)-transglycosylase (branching enzyme) Branches are created by the terminal chain segments consisting of 7 glycosyl residues to the C6-OH groups of glucose residues on another chain. Each transferred segment must be at least 11 residues. Each new branch point at least 4 residues away from other branch points.

  15. Figure 18-8 The branching of glycogen. Page 634

  16. Glycogen Breakdown Requires 3 enzymes: Glycogen phosphorylase (phosphorylase) catalyzes glycogen phosphorylysis (bond cleavage by the substitution of a phosphate group) and yields glucose-1-phosphate (G1P) Glycogen debranching enzyme removes glycogen’s branches, allowing glycogen phosphorylase to complete it’s reactions. It also hydrolyzes a(16)-linked glucosyl units to yield glucose. 92% of glycogen’s glucse residues are converted to G1P and 8% to glucose. Phosphoglucomutase converts G1P to G6P-can either go through glycolysis (muscle cells) or converted to glucose (liver).

  17. Glycogen Phosphorylase A dimer - 2 identical 842 residue subunits. Catalyzes the controlling step of glycogen breakdown. Regulated by allosteric interactions and covalent modification. Two forms of phosphorylase made by regulation Phosphorylase a- has a phosphoryl group on Ser14 in each subunit. Phosphorylase b-lacks the phosphoryl groups. Inhibitors: ATP, G6P, glucose Activator: AMP Glycogen forms a left-handed helix with 6.5 glucose residues per turn. Structure can accommodate 4-5 sugar residues only. Pyridoxal phosphate is an essential cofactor for phosphorylase. Converts glucosyl units of glycogen to G1P

  18. Figure 18-2a X-Ray structure of rabbit muscle glycogen phosphorylase. (a) Ribbon diagram of a phosphorylase b subunit. Page 628

  19. Page 630

  20. Phosphoglucomutase Converts G1P to G6P. Reaction is similar to that of phosphoglycerate mutase Difference between phosphoglycerate mutase and phosphoglucomutase is the amino acid residue to which the phosphoryl group is attached. Serine in phosphoglucomutase as opposed to His imidazole N in phosphoglycerate mutase. G1,6P occasionally dissociates from the enzyme, so catalytic amounts are necessary for activity. This is supplied by the enzyme phosphoglucokinase.

  21. Figure 18-4 The mechanism of action of phosphoglucomutase. Page 631

  22. Glycogen debranching enzyme (14) transglycosylase (glycosyl transferase) transfers a (14) linked trisaccharide unit from a limit branch to a nonreducing end of another branch. Forms a new (14) linkage with three more units available for phosphorylase. The (16) bond linking the remaining linkage is hydrolyzed by the same enzyme to yield glucose. 2 active sites on the same enzyme.

  23. Figure 18-5 Reactions catalyzed by debranching enzyme. Page 631

  24. Synthase a Normal form “active” OH OH Synthase b Requires G6P for activation “inactive” OP OP Regulation of glycogen synthesis Both synthase & phosphorylase exist in two forms. Phosphorylated at Ser residues by synthase kinase and phosphorylase kinase Pi ATP phosphoprotein phosphatase Synthase kinase ADP

  25. Regulation of glycogen synthesis AMP+, ATP-, G6P- Phosphorylase b Normal form “inactive” OH Pi OH ATP phosphorylase kinase phosphoprotein phosphatase Ca2+ ADP Phosphorylase a Independent of energy status active OP OP High [ATP] (related to high G6P) inhibits phosphorylase and stimulates glycogen synthase.

  26. Regulation of glycogen synthesis Process is also under hormonal control Adrenaline (epinephrine) can regulate glycogen synthesis/breakdown by stimulating adenylate cyclase ATP 1. External stimulus Adrenaline Adenylate cyclase cAMP cAMP + PPi 2. R2C2 cAMP dependent protein kinase [C]2 + [R-AMP]2 “inactive” “active” ADP ATP Glycogen synthase b (inactive) 3a. Glycogen synthase a (active) [C]2 ADP ATP 3b. Inactive phosphorylase kinase Active phosphorylase kinase [C]2 ADP ATP Phosphorylase a (active) Phosphorylase b (inactive)

  27. Phosphorylse b Phosphorylse a Consider the whole system Resting muscle O2 ATP respiration Glycolytic pathway pyruvate Inactive phosphorylase b, active synthase a Muscle lacks G6 Pase, Liver PFK inhibited by ATP unless F2,6P2 present Upon stress Epinephrine cAMP Synthase/phosphorylase kinase

  28. Figure 23-25 The pentose phosphate pathway. Page 863

  29. Why is the pentose phosphate pathway necessary? • ATP is the “energy currency” of cells, but cells also need reducing power. • Endergonic reactions require reducing power and ATP • Fatty acids, cholesterol, photosynthesis • NADPH and NADH are not interchangeable! • Differ only by a phosphate group at the 2’OH.

  30. O O O N N O- O- O HO OH Pi Common carrier of (H) N C-N-H2 N CH2-O-P-O-P-CH2 N (+) O HO OH NAD(P) Nicotinamide adenine dinculeotide (phosphate) (oxidized form) NADH + H+ NAD+ + 2e-

  31. O O O N O- O- O HO OH Pi Common carrier of (H) H H N C-N-H2 N CH2-O-P-O-P-CH2 N N O HO OH NAD(P) Nicotinamide adenine dinculeotide (phosphate) (reduced form) NADH + H+ NAD+ + 2e- Eº ‘ = 0.31 volt

  32. Pentose phosphate pathway • NADPH and NADH are not interchangeable! • Differ only by a phosphate group at the 2’OH. • NADH participates in utilizing the free energy of metabolite oxidation to synthesize ATP • NADPH utilizes the free energy of metaboite oxidation for biosynthesis • Difference is possible because the dehydrogenase enzymes involved in oxidative and reductive metabolism exhibit a high degree of specificity toward their respective coenzymes. • Ratios different: • [NAD+]/[NADH] is near 1000 which favors metabolite oxidation. • [NADP+]/[NADPH] is near 0.1 which favors metabolite reduction.

  33. Why is the pentose phosphate pathway necessary? • NADPH is generated by oxidation of G6P via the pentose phosphate pathway • hexose monophosphate (HMP) pathway, phosphogluconate pathway. • Alternate to glycolysis. • Produces ribose-5-phosphate (essential for nucleotide biosynthesis). Overall reaction 3G6P + 6NADP+ + 3H2O 6NADPH + 6H+ + 3CO2 + 2F6P + GP Can be considered in 3 stages

  34. Pentose phosphate pathway 3G6P + 6NADP+ + 3H2O 6NADPH + 6H+ + 3CO2 + 3Ru5P Can be divided into three stages 1. Oxidative reactions (1-3) which yield NADPH and ribulose-5-phosphate (Ru5P). 2. Isomeraization and epimeraztion reactions (4,5)-transform Ru5P to ribose-5-phosphate (R5P) or to xyulose-5-phosphate (Xu5P). R5P + 2Xu5P 3Ru5P 3. C-C bond cleavage and formation reactions (6-8)-convert 2Xu5P and R5P to 2F6P and GAP 2F6P + GAP R5P + 2Xu5P

  35. Oxidative reactions of NADPH production (1-3) • Glucose-6-phosphate dehydrogenase (G6PD)-catalyzes the net transfer of a hydride ion to NADP+ from C1 of G6P to form 6-phophoglucono--lactone. • 6-phosphoglucolactonase-increases the rate of hydrolysis of 6-phophoglucono--lactone to 6-phosphogluconate. • 6-phosphogluconate dehydrogenase catalyzes the oxidative decarboxylation of 6-phosphogluconate, a -hydroxy acid, Ru5P and CO2. (similar to isocitrate dehydrogenase)

  36. Reaction 1: The glucose-6-phosphate dehydrogenase reaction. G6PD is strongly inhibited by NADPH Page 864

  37. Reaction 2: 6-phosphoglucolactonase -2O3P-O 6-phosphoglucono--lactone 6 O 5 1 OH O 4 2 HO 3 OH 6-phosphoglucolactonase Mg2+ H2O O- O Spontaneous reaction sped up by the enzyme C H-C-OH HO-C-H H-C-OH 6-phosphogluconate H-C-OH CH2-OPO32-

  38. Reaction 3: The phosphogluconate dehydrogenase reaction. Page 864

  39. Summary of 1st stage • 3 reactions take G6P to Ru5P • G6P + NADP+ 6-phosphoglucono--lactone + NADPH • 6-phosphoglucono--lactone 6-phosphogluconate • 6-phosphogluconate + NADP+ Ru5P + CO2 + NADPH • Generates 2 molecules of NADPH for each G6P • Ru5P must be converted to R5P or Xu5P for further use.

  40. 2nd stage: isomerization and epimerization • Ru5P is converted toribose-5-phosphate (R5P)by ribulose-5-phosphate isomerase • Ru5P is converted toxyulose-5-phosphate (Xu5P)by ribulose-5-phosphate epimerase • Occur via enediolate intermediates. • R5P is an essential precursor for nucleotide biosynthesis. • If more R5P is formed than the cell needs, converted to F6P and GAP for glycolysis.

  41. NADH Page 865 ATP DNA RNA

  42. 3rd stage: carbon-carbon bond cleavage and formation reactions • Conversion of three C5 sugars to two C6 sugars and one C3 (GAP) • Catalyzed by two enzymes, transaldolase and transketolase • Mechanisms generate a stabilized carbanion which interacts with the electrophilic aldehyde center

  43. Transketolase • Transketolase catalyzes the transfer of C2 unit from Xu5P to R5P resulting in GAP and sedoheptulose-7-phosphate (S7P). • Reaction involves a covalent adduct intermediate between Xu5P and TPP. • Has a thiamine pyrophosphate cofactor that stabilizes the carbanion formed on cleavage of the C2-C3 bond of Xu5P. • The TPP ylid attacks the carbonyl group of Xu5P (C2) • C2-C3 bond cleavage results in GAP and enzyme bound 2-(1,2-dihydroxyethyl)-TPP (resonance stabilized carbanion) • The C2 carbanion attacks the aldehyde carbon of R5P forming an S7P-TPP adduct. • TPP is eliminated yielding S7P and the regenerated enzyme.

  44. CH2 CH3 Thiamine Pyrophosphate (B1) very acidic H since the electrons can delocalize into heteroatoms. H + S N N N CH3 CH2CH2O-P-P Thiazolium ring Involved in both oxidative and non-oxidative decarboxylation as a carrier of "active" aldehydes.

  45. Covalent adduct Carbanion intermediate Page 865

  46. Transketolase • Similar to pyruvate decarboxylase mechanism. • Septulose-7-phosphate (S7P) is the the substrate for transaldolase. • In a second reaction, a C2 unit is transferred from a second molecule of Xu5P to E4P (product of transaldolase reaction) to form a molecule of F6P

  47. Transaldolase • Transfers a C3 unit from S7P to GAP yielding erythrose-4-phosphate (E4P) and F6P. • Reactions occurs by aldol cleavage. • S7P forms a Schiff base with an -amino group of Lys from the enzyme and carbonyl group of S7P. • Transaldolase and Class I aldolase share a common reaction mechanism. • Both enzymes are  barrel proteins but differ in where the Lys that forms the Schiff base is located.

  48. Essential Lys residue forms a Schiff base with S7P carbonyl group • A Schiff base-stabilized C3 carbanion is formed in aldol cleavage reaction between C3-C4 yielding E4P. • The enzyme-bound resonance-stabilized carbanion adds to the carbonyl C of GAP to form F6P. • The Schiff base hydrolyzes to regenerate the original enzyme and release F6P Page 866

  49. Figure 23-31 Summary of carbon skeleton rearrangements in the pentose phosphate pathway. Page 867

  50. Control of Pentose Phosphate Pathway Principle products are R5P and NADPH. Transaldolase and transketolase convert excess R5P into glycolytic intermediates when NADPH needs are higher than the need for nucleotide biosynthesis. GAP and F6P can be consumed through glycolysis and oxidative phosphorylation. Can also be used for gluconeogenesis to form G6P 1 molecule of G6P can be converted via 6 cycles of PPP and gluconeogenesis to 6 CO2 molecules and generate 12 NADPH molecules. Flux through PPP (rate of NADPH production) is controlled by the glucose-6-phosphate dehydrogense reaction. G6PDH catalyzes the first committed step of the PPP.

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