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

2. Figure 23-25The 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 18-1aStructure of glycogen. (a) Molecular formula. Page 627

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

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

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

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

9. Figure 18-6Reaction 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 18-7Reaction catalyzed by glycogen synthase. Page 633 O

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 18-8The branching of glycogen. Page 634

16. Glycogen Breakdown Requires 3 enzymes: 1.Glycogen phosphorylase (phosphorylase) catalyzes glycogen phosphorylysis (bond cleavage by the substitution of a phosphate group) and yields glucose-1- phosphate (G1P) 2.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. 3.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 18-2aX-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 18-4The 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 18-5Reactions catalyzed by debranching enzyme. Page 631

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

25. Regulation of glycogen synthesis Phosphorylase b Normal form “inactive” Phosphorylase a Independent of energy status active OH OP ATP ADP Pi phosphorylase kinase phosphoprotein phosphatase AMP+, ATP-, G6P- Ca 2+ 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 1. External stimulus Adrenaline Adenylate cyclase ATP cAMP + PPi 2. R 2 C 2 [C] 2 + [R-AMP] 2 cAMP dependent protein kinase cAMP “inactive” “active” 3a. Glycogen synthase a (active) Glycogen synthase b (inactive) ATP ADP [C] 2 3b. Inactive phosphorylase kinase Active phosphorylase kinase ATP ADP [C] 2 ATP ADP Phosphorylase b (inactive) Phosphorylase a (active)

27. Consider the whole system Resting muscle Glycolytic pathway pyruvate ATP cAMP O2O2 respiration Inactive phosphorylase b, active synthase a Muscle lacks G6 Pase, Liver PFK inhibited by ATP unless F2,6P2 present Upon stress Epinephrine Synthase/phosphorylase kinase Phosphorylse bPhosphorylse a

28. Figure 23-25The 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. Common carrier of (H) NAD(P) Nicotinamide adenine dinculeotide (phosphate) (oxidized form) O N N N N O OH HO O- O O O OH HO CH 2 -O-P-O-P-CH 2 N C-N-H 2 (+) PiPi NAD + + 2e - NADH + H +

31. Common carrier of (H) NAD(P) Nicotinamide adenine dinculeotide (phosphate) (reduced form) O N N N N O OH HO O- O O O OH HO CH 2 -O-P-O-P-CH 2 N C-N-H 2 PiPi H H 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). 3G6P + 6NADP + + 3H 2 O Overall reaction 6NADPH + 6H + + 3CO 2 + 2F6P + GP Can be considered in 3 stages

34. Pentose phosphate pathway Can be divided into three stages 1. Oxidative reactions (1-3) which yield NADPH and ribulose-5- phosphate (Ru5P). 3G6P + 6NADP + + 3H 2 O 6NADPH + 6H + + 3CO 2 + 3Ru5P 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) 1.Glucose-6-phosphate dehydrogenase (G6PD)-catalyzes the net transfer of a hydride ion to NADP + from C1 of G6P to form 6-phophoglucono-  -lactone. 2.6-phosphoglucolactonase-increases the rate of hydrolysis of 6-phophoglucono-  -lactone to 6- phosphogluconate. 3.6-phosphogluconate dehydrogenase catalyzes the oxidative decarboxylation of 6-phosphogluconate, a  - hydroxy acid, Ru5P and CO 2. (similar to isocitrate dehydrogenase)

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

37. Reaction 2: 6-phosphoglucolactonase O 1 O -2 O 3 P-O OH HO OH 2 3 4 5 6 6-phosphoglucono-  -lactone 6-phosphoglucolactonase Mg 2+ H2OH2O C H - C - OH HO - C - H H - C - OH CH 2 - OPO 3 2- H - C - OH O O-O- 6-phosphogluconate Spontaneous reaction sped up by the enzyme

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 + CO 2 + 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 to ribose-5-phosphate (R5P) by ribulose-5-phosphate isomerase Ru5P is converted to xyulose-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. Page 865 NADH 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. 1.The TPP ylid attacks the carbonyl group of Xu5P (C2) 2.C2-C3 bond cleavage results in GAP and enzyme bound 2-(1,2- dihydroxyethyl)-TPP (resonance stabilized carbanion) 3.The C2 carbanion attacks the aldehyde carbon of R5P forming an S7P-TPP adduct. 4.TPP is eliminated yielding S7P and the regenerated enzyme.

44. Thiamine Pyrophosphate (B1) Thiazolium ring CH 3 CH 2 CH 3 CH 2 CH 2 O-P-P H S N N N + very acidic H since the electrons can delocalize into heteroatoms. Involved in both oxidative and non-oxidative decarboxylation as a carrier of "active" aldehydes.

45. Page 865 Covalent adduct Carbanion intermediate

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. Page 866 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

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

50. Control of Pentose Phosphate Pathway 1.Principle products are R5P and NADPH. 2.Transaldolase and transketolase convert excess R5P into glycolytic intermediates when NADPH needs are higher than the need for nucleotide biosynthesis. 3.GAP and F6P can be consumed through glycolysis and oxidative phosphorylation. 4.Can also be used for gluconeogenesis to form G6P 5.1 molecule of G6P can be converted via 6 cycles of PPP and gluconeogenesis to 6 CO 2 molecules and generate 12 NADPH molecules. 6.Flux through PPP (rate of NADPH production) is controlled by the glucose-6-phosphate dehydrogense reaction. 7.G6PDH catalyzes the first committed step of the PPP.