Lecture 23 –Quiz next Mon. on Pentose Phosphate Pathway –Metabolic regulation and control of glycolysis/gluconeogenesis

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Lecture 23 Quiz next Mon. on Pentose Phosphate Pathway Metabolic regulation and control of glycolysis/gluconeogenesis Slide 2 Hydrolytic reactions bypass PFK and Hexokinase Page 602 Instead of generating ATP by reversing the glycolytic reactions, FBP and G6P are hydrolyzed to release P i in an exergonic reaction. Slide 3 Page 848 Slide 4 Gluconeogenesis Glucose + 2ADP + 2P i + 2NAD + 2 Pyruvate + 2ATP + 2NADH + 4H + + 2H 2 O Net reaction 2ADP + 2GDP + 4P i 2ATP + 2GTP + 4H 2 O Glycolysis 2 Pyruvate + 4ATP + 2GTP 2NADH + 4H + + 6H 2 O Glucose + 4ADP +2GDP + 6P i + 2NAD + Slide 5 Control Points in Glycolysis Slide 6 1st reaction of glycolysis ( G = -4 kcal/mol) OH 1 O HO OH HO OH * 2 3 4 5 6 Glucose OH 1 O -2 O 3 P-O OH HO OH * 2 3 4 5 6 ATP ADP Glucose-6-phosphate (G6P) Hexokinase (HK) I, II, II Muscle(II), Brain (I) Mg 2+ Glucokinase (HK IV) in liver Slide 7 Regulation of Hexokinase Glucose-6-phosphate is an allosteric inhibitor of hexokinase. Levels of glucose-6-phosphate increase when downstream steps are inhibited. This coordinates the regulation of hexokinase with other regulatory enzymes in glycolysis. Hexokinase is not necessarily the first regulatory step inhibited. Slide 8 Types of regulation 1.Availability of substrate Glucokinase (K M 12 mM) vs. HK (K M = 0.01 - 0.03 mM) 2.Compartmentalization -Brain vs. Liver vs. Muscle (type I mitochondrial membrane, type II cytoplasmic) 3.Allosteric regulation - feedback inhibition by G-6-P, overcome by Pi in type I (Brain/ mitochondrial controlled by Pi levels) 4.Hormonal regulation. Liver has HK as fetal tissue. Changes to glucokinase after about 2 weeks. If there is no dietary carbohydrate, no glucokinase. Must have both insulin and carbohydrates to induce. Slide 9 2 places where there is no net reaction 1.ATP + F-6-P F-1,6-P 2 + ADP 2. F-1,6-P 2 F-6-P + P i PFK F-phosphatase Mg 2+ Net: ATP ADP + P i + heat Similar reaction occurs with hexokinase and G-6-phosphatase. Generally regulated so this does not occur (futile cycle). May function in hibernating animals to generate heat. Slide 10 Primary regulation - reciprocal with energy charge Enzyme+- HexokinaseG-6-P PFKP i, ADP, AMP, F-6-P, F-2,6-P 2 ATP, citrate, NADH F-6- phosphatas e ATPAMP, F-2,6- P 2 Pyruvate kinase K +, AMP, F- 2,6-P 2 ATP, acetyl- CoA, cAMP Pyruvate carboxylase Acetyl-CoA Slide 11 Major regulation is through energy charge ATP Gluconeogenesis Glycolysis ADP Same reactions make AMP or ADP (primarily in lipid and nucleotide metabolism) AMP + ATP 2 ADP Adenylate kinase [ATP] +1/2[ADP] [AMP] + [ADP] + [ATP] Energy charge 1.0 = 100% ATP Body generally likes it close to 0.9 0.5 = 100% ADP 0 = 100% AMP Slide 12 Control Points in Glycolysis Slide 13 Primary regulation - reciprocal with energy charge Enzyme+- HexokinaseG-6-P PFKP i, ADP, AMP, F-6-P, F-2,6-P 2 ATP, citrate, NADH F-6- phosphatas e ATPAMP, F-2,6- P 2 Pyruvate kinase K +, AMP, F- 2,6-P 2 ATP, acetyl- CoA, cAMP Pyruvate carboxylase Acetyl-CoA Slide 14 Regulation of PhosphoFructokinase (PFK-1) PKF-1 has quaternary structure Inhibited by ATP and Citrate Activated by AMP and Fructose-2,6- bisphosphate Regulation related to energy status of cell. Slide 15 PFK-1 regulation by adenosine nucleotides ATP is substrate and inhibitor. Binds to active site and allosteric site on PFK. Binding of ATP to allosteric site increase K m for ATP AMP and ADP are allosteric activators of PFK. AMP relieves inhibition by ATP. ADP decreases K m for ATP Glucagon (a pancreatic hormone) produced in response to low blood glucose triggers cAMP signaling pathway that ultimately results in decreased glycolysis. Slide 16 Effect of ATP on PFK-1 Activity Slide 17 Effect of ADP and AMP on PFK-1 Activity Slide 18 Regulation of PFK by Fructose-2,6-bisphosphate Fructose-2,6-bisphosphate is an allosteric activator of PFK in eukaryotes, but not prokaryotes Formed from fructose-6-phosphate by PFK-2 Degraded to fructose-6-phosphate by fructose 2,6- bisphosphatase. In mammals the 2 activities are on the same enzyme PFK-2 inhibited by Pi and stimulated by citrate Slide 19 Fructose-2,6-bisphosphate can override Energy charge Produced when [glucose] is high but need glycolysis for anabolic role. When glucose is needed by the brain (about 120 g/day via diet or other tissues) Glucose F-6-P F-1,6-P 2 F-2,6-P 2 F-1,6-Pase F-2,6-Pase PFK-2 PFK-1 Bifunctional enzyme ATP - F-6-P + AMP + F-2,6-P2 + Citrate- PEP - AMP- F-2,6-P 2 - cAMP + NTP + 3 PGA - cAMP - Citrate+ Slide 20 Glucagon Regulation of PFK-1 in Liver PFK-1 normally inhibited by ATP G-Protein mediated cAMP signaling pathway Induces protein kinase A that activates phosphatase activity and inhibits kinase activity Results in lower F-2,6-P levels decrease PFK-1 activity (less glycolysis) Slide 21 PFK-2 1.Serves to override ATP inhibition and promote glycolysis once intermediates build up [citrate] [PEP][GAP] 2.Block PFK-2 activity with high [NTP] by stimulating F- 2,6-Pase This will break down F-2,6-P2 and restores energy charge regulation. cAMP is the hormonal control. The presence of cAMP is indicative of low blood sugar (glucagon) stimulates F- 2,6-Pase to increase F-6-P formtion for gluconeogenesis (cAMP also inhibits Pyruvate Kinase). Slide 22 Regulation of Pyruvate Kinase Allosteric enzyme Activated by Fructose-1,6-bisphosphate (example of feed-forward regulation) Inhibited by ATP When high fructose 1,6-bisphosphate present plot of [S] vs Vo goes from sigmoidal to hyperbolic. Increasing ATP concentration increases Km for PEP. In liver, PK also regulated by glucagon. Protein kinase A phosphorylates PK and decreases PK acitivty. Slide 23 Pyruvate Kinase Regulation Slide 24 Deregulation of Glycolysis in Cancer Cells Glucose uptake and glycolysis is 10X faster in solid tumors than in non-cancerous tissues. Tumor cells initally lack connection to blood supply so limited oxygen supply Tumor cells have fewer mitochondrial, depend more on glycolysis for ATP Increase levels of glycolytic enzymes in tumors (oncogene Ras and tumor suppressor gene p53 involved) Slide 25 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. Slide 26 Figure 18-1aStructure of glycogen. (a) Molecular formula. Page 627 Slide 27 Figure 18-1bStructure of glycogen. (b) Schematic diagram illustrating its branched structure. Page 627 Slide 28 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 Slide 29 Glycogen biosynthesis 3 enzymes catalyze the steps involved in glycogen synthesis: UDP-glucose pyrophosphorylase Glycogen synthase Glycogen branching enzyme Slide 30 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 Slide 31 Figure 18-6Reaction catalyzed by UDPglucose pyrophosphorylase. Page 633 Slide 32 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. Slide 33 Glycogen synthase In this step, the glucosyl unit of UDP-glucose (UDPG) is transferred to the C4-OH group of one of glycogens 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 Slide 34 Figure 18-7Reaction catalyzed by glycogen synthase. Page 633 O Slide 35 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. Slide 36 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. Slide 37 Figure 18-8The branching of glycogen. Page 634 Slide 38 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 glycogens branches, allowing glycogen phosphorylase to complete its reactions. It also hydrolyzes a(16)-linked glucosyl units to yield glucose. 92% of glycogens 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). Slide 39 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- h

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