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3 - Lecture Lecture 3 CARBOHYDRATE METABOLISM(Part1...

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Unformatted text preview: Lecture Lecture 3 CARBOHYDRATE METABOLISM (Part1) GLYCOLYSIS Ch Chapter 17 (sections 1,2,3,5 and 23 (section 4) 17 23 4) in in Voet & Voet) BCH3120 General Intermediary Metabolism General Lecturer: Alfred Aziz, PhD Alfred Aziz PhD Nutrition Research Division, Health Canada e-mail: [email protected] [email protected] 12, 2011 Outline Outline 1. 2. 3. 4. 5. Introduction Glycolysis- concepts and detailed reactions ti Regulation of glycolysis Catabolism of other carbohydrates through glycolysis Alternative glycolytic pathway for rapidly proliferating cells 1. Metabolism is the Sum of Cellular Reactions Major Catabolic Pathways Major Catabolic Pathways Glycogen and starch and starch Proteins Glucose Amino Acids ATP Pyruvate Acetyl-CoA Oxaloacetate GTP NH4+ NADH ATP QH2 Triglycerides Fatty Acids Metabolism Proceeds by Discrete Steps Major Concepts Major Concepts 1. 2. 3. Glycolysis is a series of reactions in which one molecule of glucose is converted to two molecules of pyruvate yielding two molecules of pyruvate, yielding biologically biologically useful energy in the form of ATP and NADH Carbohydrates other than glucose also feed into the glycolytic pathway Pyruvate has three possible fates after glycolysis Major Concepts… continued Major Concepts continued 4. 5. 6. A cell’s ability to regenerate NAD+ is critical for continued glycolysis Fermentation is used in the th production of common foods, beverages and industrial chemicals beverages and industrial chemicals. It can also occur in muscle! The pentose phosphate pathway is The pentose phosphate pathway is an an alternative pathway for glucose oxidation 2. Glycolysis Glycolysis A series of reactions in which one molecule of glucose is degraded to two molecules of pyruvate, yielding biologically useful energy in the pyruvate, form of ATP and NADH (Animals) (Plants) Net Reaction of Glycolysis Net Reaction of Glycolysis Glucose + 2 ADP + 2 NAD+ + 2 Pi 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O ATP NADH Animations: How Glycolysis Works Gl 1. An extremely simple overview: http://highered.mcgraw-hill.com/sites/0072507470/student_view0/chapter25/animation__how_glycolysis_works.html 2. A molecular perspective: http://www.iubmb-nicholson.org/swf/glycolysis.swf The Enzymatic Reactions of Glycolysis ‘‘lysis’ step l Fig. 17-3 Fig. 17-3 • Net yield of ATP per molecule of glucose is 4-2 = 2 4• NADH yield is 2 Take note! Nota bene! Take note! Nota bene! Coupling of exergonic reaction (glucose oxidation) to endergonic reaction (ADP Pi reaction (ADP + Pi -> ATP) allows ATP) allows Substrate Substrate Level Phosphorylation Glycolysis releases only a small fraction small of the energy contained in glucose. Most of the E is extracted in of Oxidative Phosphorylation Oxidative Phosphorylation 1. Phosphorylation of Glucose Gl • An activation step • Hexokinase: Hexokinase: • Normally glucose, but also fructose and mannose Like many kinases it requires Mg • Like many kinases, it requires Mg++ (MgATP) • Found in all cells in various isoforms • Liver contains a functionally unique isoform, ‘glucokinase’ isoform, ‘glucokinase’ Hexokinase: Conformation changes on binding glucose changes on binding glucose (glu, purple; C atoms in large lobe are gray, in small lobe green; N and O are blue and red respectively) Fig. 17-4 2. Conversion of Glucose 2. Conversion of Glucose 6-P to Fructose 6-P to Fructose • • • Again Mg++ required, like most enzymes in glycolysis Reversible Rearrangement of carbonyl and hydroxyl groups at C1 and C2 3. 3. Phosphorylation of Fructose 6-P to Fructose 1,6 Bisphosphate 6• • • • Second of the two priming reactions Why do you think the E is called PFK-1 ? PFKEssentially irreversible in cells MAJOR REGULATORY step (more on this later) – a sensor of ATP, ADP and AMP ketose aldose aldose 4. Cleavage of Fructose 1,6 Bisphosphate (the ‘lysis’ step) Cleavage of Fructose Bisphosphate step) • Even though standard free energy change is high and positive, concentrations of reactants and products make the forward concentrations of reactants and products make the forward reaction reaction possible. Step 5: Triose phosphate isomerase G3P is released first from Aldolase, then DHAP Ald th DHAP BUT, only G3P can continue DHAP DHAP is readily converted to G3P so G3P that its energy can be further metabolized too Question: Question: If If you labeled carbon#1 14 with 14C or 3H , could you specify which carbon in G3P would be radioactive? Answer: Answer: Carbon#3 of G3P (50% of the C3s in G3P would be labeled) Payoff Phase • ATP • NADH + H+ 6. Oxidation of G3P to 1,3 Bisphosphoglycerate Reduction of NAD+ occurs through enzymatic transfer of a hydride ion ( :H- ) from aldehyde group to NAD+ ring ** Cells have relatively low concentrations of NAD+; glycolysis stops if there is insufficient oxidized NAD+ 7. 7. • transfer of high energy transfer of high energy phosphoryl phosphoryl group to ADP:substrateADP:substrate-level phosphorylation • Note: E name is for reverse reaction (it catalyses reaction in catalyses reaction in both both directions); the latter occurs in gluconeogenesis gluconeogenesis. • Phosphoglycerate Kinase (PKG) Phosphoryl transfer from 1,3 bisphosphoglycerate to ADP Note: exergonic reaction 7 pulls endergonic reaction 6 forward. G = -18.5 kJ/mol Fig. 17-16 “Substrate“Substrate-level phosphorylation” 8. 8. Conversion of 3-phosphoglycerate to 32-phosphoglycerate • phosphoryl group moves to C2 • 2, 3 bisphosphoglycerate is a temporary intermediate 8. 8. 1. Formation of ES complex 2. Transfer of phosphoryl group from enzyme to substrate substrate 3. Rephosphorylation of enzyme by the other phosphoryl phosphoryl group of the substrate 4. Fig. 17-18 Conversion of 3-phosphoglycerate to phosphoglycerate to 2-phosphoglycerate Release of product of product Mg2+ 9. Dehydration of 2-phosphoglycerate to PEP 2• PEP has a very high standard free energy of hydrolysis very -61.9 kJ/mol (over 3x higher than 2-phosphoglycerate) 2• created by the removal of water 10 Phosphoryl group transfer to ADP 10. Phosphoryl group transfer to ADP • Last step of glycolysis • “Substrate level phosphorylation” • Essentially irreversible • Important site of control The Final Product: Initial, and only temporary Main form in cells Reviewing the Pathway ‘lysis’ step step Reviewing the Pathway • Net yield of ATP per molecule of glucose is 4-2 = 2 4• NADH yield is 2 Catabolic Catabolic Fates of Pyruvate, the product of Glycolysis: Reviewing the Pathway 3. Regulation of Glycolysis Regulation of Glycolysis 1. 2. 3. 4. Regulation of hexose transporters Regulation of hexokinase Regulation of Phosphofructokinase-1 Regulation of Pyruvate kinase of Pyruvate kinase Family of glucose transporters Family of glucose transporters Insulin regulates transport of glucose in muscles and adipose tissue via GLUT4 Regulation of hexokinase Regulation of hexokinase 4 different isozymes: I, II, III, and IV Hexokinase IV = glucokinase Hexokinase vs Most tissues Low Km (0.1mM) Low Vmax Inhibition by G6P Glucokinase Liver and β-cells High Km (5-10mM) High Vmax No inhibition by G6P Regulation of glucokinase Regulation of glucokinase Fructose 1-phosphate Fructose 6-phosphate Regulation of Phosphofructokinase-1 Inhibitor ATP ADP (plant) Citrate (TCA cycle) (TCA cycle) Low pH Activator AMP ADP (mammals) Fructose 2,6-bisphosphate Regulation of Phosphofructokinase-1 by ATP and AMP Fructose 2,6-bisphosphate metabolism Effect of glucagon on glycolysis Protein Kinase A Regulation of Pyruvate Kinase Regulation of Pyruvate Kinase Summary of the Metabolic Regulation of Glycolysis Activation Inhibition 4. Carbohydrates other than glucose enter the glycolytic glucose enter the glycolytic pathway Dietary starch or sucrose: Glucose Dietary sucrose fructose Fructose Dietary sucrose or fructose: Fructose Dietary lactose: Galactose Tissue glycogen stores: Glucose Glucose EM micrograph of glycogen from rat skeletal muscle. is one granule, which is composed of several spherical glycogen molecules (). polysaccharides disaccharides … into the Preparatory Phase of Glycolysis Triose phosphate isomerase Glu Glu-6-P UDP UDP-galactose is converted to converted to UDP-glucose by UDP-glucose 4 epimerase (an enzyme in which sequential reactions first generate NADH + H and then consume NADH + H) Note the error in this diagram the error this diagram (from Lehninger text) Glycolysis NADH + H NAD+ Corrected Lactase deficiency Lactase deficiency The inability to digest significant amounts of lactose, the major sugar in milk. Caused by a shortage of the enzyme lactase, produced in cells that line the small intestine. Lactase hydrolyzes lactose into glucose and galactose. Not all people deficient in lactase have the symptoms commonly associated with lactose intolerance, but those who do are ‘lactose intolerant’. Nausea, cramps, bloating, gas, and diarrhea. Primary lactase deficiency develops gradually. After about age 2, the body produces less lactase though symptoms are usually not apparent until produces less lactase, though symptoms are usually not apparent until much later. Secondary lactase deficiency occurs when injury to the small intestine or certain digestive diseases reduce the amount of lactase a person produces. E.g., celiac disease, inflammatory bowel disease, and Crohn’s disease. celiac disease inflammatory bowel disease and Crohn disease Primary lactase deficiency affects up to 80% of African Americans, 80-100% of American Indians, and 90-100% of Asian Americans are lactose intolerant. Least common among people of northern European descent. 5. Alternative Glycolytic Pathway in Rapidly Pathway in Rapidly Proliferating Cells Normal cells catabolize glucose by oxidative phosphorylation Rapidly proliferating cells, such as cancerous cells metabolize glucose by aerobic glycolysis with production of lactate (Warburg effect) favouring conversion of glucose into macromolecules needed for new cells Vander Heiden et al., Science 2010; 329:1492-99 Alternative Glycolytic Pathway in Rapidly Proliferating Cells in Rapidly Proliferating Cells (cont’d) The isoform of pyruvate kinase determines the preferential metabolic pathway for glucose: PKM1 PKM2 oxidative phosphorylation aerobic glycolysis All cancer cells express PKM2 Paradox: Low pyruvate kinase activity important for cell proliferation Vander Heiden et al., Science 2010; 329:1492-99 PKM2 expressing cells have low pyruvate kinase activity Vander Heiden et al., Science 2010; 329:1492-99 So, how is pyruvate formed in rapidly proliferating cells in the presence of low pyruvate kinase activity ??? Normal pathway using PKM1 splice isoform Pyruvate Kinase Alternative pathway using PKM2 splice isoform Phosphoglycerate mutase l Unknown enzyme Phosphoglycerate mutase kinase mutase kinase Take Home Messages Take Home Messages Glycolysis converts 1 molecule of glucose to 2 molecules of pyruvate There is a net production of 2 ATP molecules Glycolysis has 3 points of regulation (HK or GK, PFK-1 and PK) Carbohydrates other than glucose enter the glycolytic pathway th An alternative glycolytic pathway that produces pyruvate through a pyruvate kinase independent reaction exists in rapidly proliferating cells reaction exists in rapidly proliferating cells Th Thank you for your attention ...
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