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Lecture 24 – Quiz Mon. on Pentose Phosphate Pathway – Glycogen regulation – Quiz next Fri. on TCA cycle Page 863 Figure 23-25 The pentose phosphate pathway. 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. Page 627 Figure 18-1a Structure of glycogen. (a) Molecular formula. Page 627 Figure 18-1bStructure of glycogen. (b) Schematic diagram illustrating its branched structure. Glycogen is an efficient storage form UDP-glucose G-6-P G-1-P + UTP UDP + ATP Glycogen + UDP + Pi UTP + ADP Net: 1 ATP required 90% 1,4 residues Glycogen + Pi 10% 1,6 residues Glycogen G-1-P G-6-P glucose 1.1 ATP/38 ATP so, about a 3% loss, therefore it is about 97% efficient for storage of glucose Glycogen biosynthesis 3 enzymes catalyze the steps involved in glycogen synthesis: UDP-glucose pyrophosphorylase Glycogen synthase Glycogen branching enzyme Glycogen biosynthesis MgATP Glucose HK MgADP [G-1,6-P2] G-6-P F-6-P G-1-P phosphoglucomutase PGI The hydrolysis of pyrophosphate to inorganic phosphate is highly exergonic and is catalyzed by inorganic pyrophosphatase PPase UTP G-1-P PPi UDP-Glucose Pyrophosphorylase 2Pi Page 633 Figure 18-6 Reaction catalyzed by UDP–glucose pyrophosphorylase. 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. 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 Figure 18-7 Reaction catalyzed by glycogen synthase. Page 633 O 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. Glycogen branching Catalyzed by amylo (1,41,6)-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. Page 634 Figure 18-8 The branching of glycogen. Glycogen Breakdown Requires 3 enzymes: 1. Glycogen phosphorylase (phosphorylase) catalyzes glycogen phosphorylysis (bond cleavage by the substitution of a phosphate group) and yields glucose-1phosphate (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). 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 Page 628 Figure 18-2a X-Ray structure of rabbit muscle glycogen phosphorylase. (a) Ribbon diagram of a phosphorylase b subunit. Page 630 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. Page 631 Figure 18-4 The mechanism of action of phosphoglucomutase. Glycogen debranching enzyme (14) transglycosylase (glycosyl transferase) transfers a (14) linked trisaccharide unit from a limit branch to a nonreducing end of another branch. Forms a new (14) linkage with three more units available for phosphorylase. The (16) bond linking the remaining linkage is hydrolyzed by the same enzyme to yield glucose. 2 active sites on the same enzyme. Page 631 Figure 18-5 Reactions catalyzed by debranching enzyme. 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” Pi OH OH phosphoprotein phosphatase ATP Synthase kinase ADP Synthase b Requires G6P for activation “inactive” OP OP Regulation of glycogen synthesis AMP+, ATP-, G6PPhosphorylase b Normal form “inactive” Pi phosphoprotein phosphatase OH OH ATP phosphorylase kinase Ca2+ ADP Phosphorylase a OP Independent of energy status OP active High [ATP] (related to high G6P) inhibits phosphorylase and stimulates glycogen synthase. 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 cAMP cyclase 2. R2C2 cAMP dependent protein kinase “inactive” [C]2 + [R-AMP]2 “active” ATP ADP Glycogen synthase b (inactive) 3a. Glycogen synthase a [C]2 (active) ATP 3b. Inactive phosphorylase kinase Phosphorylase b (inactive) cAMP + PPi ADP Active phosphorylase kinase [C]2 ATP ADP Phosphorylase a (active) Consider the whole system Resting muscle Glycolytic pathway pyruvate O2 respiration ATP Inactive phosphorylase b, active synthase a Muscle lacks G6 Pase, Liver PFK inhibited by ATP unless F2,6P2 present Upon stress Epinephrine cAMP Phosphorylse b Synthase/phosphorylase kinase Phosphorylse a Page 863 Figure 23-25 The pentose phosphate pathway. 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. Common carrier of (H) O N N O N N O HO C-N-H2 O CH2-O-P-O-P-CH2 O O- OOH HO N(+) OH Pi NAD(P) Nicotinamide adenine dinculeotide (phosphate) (oxidized form) NAD+ + 2e- NADH + H+ Common carrier of (H) H H N N O N N O HO C-N-H2 O CH2-O-P-O-P-CH2 O O- OOH O HO N OH Pi NAD(P) Nicotinamide adenine dinculeotide (phosphate) (reduced form) NADH + H+ NAD+ + 2e- Eº ‘ = 0.31 volt 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. 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 Can be considered in 3 stages 6NADPH + 6H+ + 3CO2 + 2F6P + GP Pentose phosphate pathway Can be divided into three stages 1. Oxidative reactions (1-3) which yield NADPH and ribulose-5phosphate (Ru5P). 3G6P + 6NADP+ + 3H2O 6NADPH + 6H+ + 3CO2 + 3Ru5P 2. Isomeraization and epimeraztion reactions (4,5)-transform Ru5P to ribose-5-phosphate (R5P) or to xyulose-5phosphate (Xu5P). 3Ru5P R5P + 2Xu5P 3. C-C bond cleavage and formation reactions (6-8)-convert 2Xu5P and R5P to 2F6P and GAP R5P + 2Xu5P 2F6P + GAP Oxidative reactions of NADPH production (1-3) 1. 2. 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 6phosphogluconate. 6-phosphogluconate dehydrogenase catalyzes the oxidative decarboxylation of 6-phosphogluconate, a hydroxy acid, Ru5P and CO2. (similar to isocitrate dehydrogenase) Reaction 1: The glucose-6-phosphate dehydrogenase reaction. Page 864 G6PD is strongly inhibited by NADPH Reaction 2: 6-phosphoglucolactonase -2O P-O 3 6-phosphoglucono--lactone 6 O 5 OH 4 1 2 HO O 3 OH H2O O OC H-C-OH 6-phosphoglucolactonase Mg2+ Spontaneous reaction sped up by the enzyme HO-C-H H-C-OH H-C-OH 6-phosphogluconate CH2-OPO32- Page 864 Reaction 3: The phosphogluconate dehydrogenase reaction. 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. 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. Page 865 NADH ATP DNA RNA 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 Transketolase • • • 1. 2. 3. 4. 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-(1,2dihydroxyethyl)-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. Thiamine Pyrophosphate (B1) very acidic H since the electrons can delocalize into heteroatoms. H CH2 N CH3 N + N S CH3 CH2CH2O-P-P Thiazolium ring Involved in both oxidative and non-oxidative decarboxylation as a carrier of "active" aldehydes. Covalent adduct Page 865 Carbanion intermediate 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 Transaldolase • • • • • Transfers a C3 unit from S7P to GAP yielding erythrose-4phosphate (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. • 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 resonancestabilized carbanion adds to the carbonyl C of GAP to form F6P. Page 866 • • The Schiff base hydrolyzes to regenerate the original enzyme and release F6P Page 867 Figure 23-31 Summary of carbon skeleton rearrangements in the pentose phosphate pathway. 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 CO2 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.