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General Biochemistry Part II-a Prof. Dr. E. Van Driessche Prof. Dr. S. Beeckmans [email protected] Protein Chemistry Lab Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, room E.5.23 I. The foundations of biochemistry 1. 2. The cell and subcellular organelles Short overview of biomolecules: proteins, nucleic acids, lipids, polysaccharides, and their building blocks Fundamentals of energy and energy transfer 3. Prof. Dr. Edilbert Van Driessche II. Metabolism and its regulation 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Strategies to regulate metabolic pathways Glycolysis and gluconeogenesis Pentose-phosphate pathway and Calvin cycle Glycogen metabolism Fatty acid degradation (-oxidation and -oxidation) Fatty acid biosynthesis and ketone bodies Pyruvate dehydrogenase, citric acid cycle and anaplerotic reactions Amino acid degradation and urea cycle Nitrogen metabolism: biosynthesis of amino acids, nucleotides and related molecules Oxidative phosphorylation and ATP synthesis Photosynthesis (light reactions) III. Intra- and intercellular organization 15. 16. 17. 18. 19. 20. Biomembranes (including transport systems) The cytoskeleton Metabolons: physical organization of metabolic pathways Protein trafficking Protein degradation Integration of metabolism: from molecules to cells to organs Prof. Dr. Edilbert Van Driessche Major pathways of glucose utilization Glycolysis From Greek: glycos (sugar) and lysis (dissolution) Prof. Dr. Edilbert Van Driessche Definition: glycolysis is the sequence of reactions that convert glucose into pyruvate with the concomitant production of ATP and NADH. History of glycolysis 1897 − Hans Buchner & Edward Buchner: ● sucrose added to cell-free extracts of yeast results in the formation of alcohol. fermentation can occur outside living cells. 1905 − Arthur Harden & William Young: ● glucose added to yeast extract results in an immediate start of the fermentation, but the rate of fermentation rapidly decreases unless inorganic phosphate is added ● inorganic phosphate is incorporated into sugar phosphates ● yeast juice contains at least 2 kinds of substances necessary for fermentation, i.e. “zymase” and “cozymase”. Prof. Dr. Edilbert Van Driessche yeast juice 50°C dialysis inactive inactive mix both: active juice Today: zymases are enzymes, cozymases are metal ions, ATP, ADP, coenzymes (NAD, FAD, ...), magnesium, ..... 1940 − Gustav Embden, Otto Mayerhof, Otto Warburg, Carl Neuberg, Jacob Parnas, and Gerty & Carl Cori: elucidation of the glycolytic pathway Prof. Dr. Edilbert Van Driessche Glycolysis: two phases Prof. Dr. Edilbert Van Driessche Prof. Dr. Edilbert Van Driessche Prof. Dr. Edilbert Van Driessche Fate of pyruvate under anaerobic conditions: fermentations Prof. Dr. Edilbert Van Driessche Aim: regeneration of NAD+ Gluconeogenesis: “formation of new sugar” A universal pathway Glycolysis and gluconeogenesis both take place in the same compartment, i.e. the cytoplasm Prof. Dr. Edilbert Van Driessche need for rigorous regulation Carbohydrate synthesis from simple precursors Opposing pathways glycolysis and gluconeogenesis Glycolysis and gluconeogenesis share many steps Remember: Prof. Dr. Edilbert Van Driessche 3 steps in glycolysis are irreversible bypases using seperate sets of enzymes are required Prof. Dr. Edilbert Van Driessche Prof. Dr. Edilbert Van Driessche Alternative paths from pyruvate to PEP From pyruvate to phosphoenolpyruvate Mitochondria: PCX Pyruvate + HCO3− + ATP Oxaloacetate + NADH + H+ Cytoplasm: oxaloacetate + ADP + Pi mMDH cMDH Malate + NAD+ Prof. Dr. Edilbert Van Driessche Oxaloacetate + GTP malate + NAD+ oxaloacetate + NADH + H+ PEP-CK Pyruvate + ATP + GTP + HCO3− phosphoenolpyruvate + CO2 + GDP PEP + ADP + GDP + Pi + CO2 ΔG’° = +0.9 kJ/mol ΔG = −25 kJ/mol PCX MDH PEP-CK pyruvate carboxylase malate dehydrogenase phosphoenolpyruvate carboxykinase Synthesis of PEP from pyruvate Prof. Dr. Edilbert Van Driessche Reaction in the mitochondria Reaction in the cytoplasm Regulation of glycolysis and gluconeogenesis Remember: Glycolysis and gluconeogenesis are both occurring in the cytoplasm The one is not simply the reversal of the other 7 of the glycolytic reactions are freely reversible and the enzymes catalyzing them also operate in gluconeogenesis 3 reactions of glycolysis are essentially irreversible (large negative G’) Prof. Dr. Edilbert Van Driessche in gluconeogenesis these reactions are bypassed Glycolysis: Gluconeogenesis: hexokinase phosphofructokinase-1 pyruvate kinase glucose-6-phosphatase fructose-1,6-bisphosphatase pyruvate carboxylase PEP carboxykinase At each of these points, when glycolysis and gluconeogenesis would simultaneously operate, ATP would be consumed without accomplishing any chemical or biological work. Example: ATP + fructose-6-phosphate PFK-1 fructose-1,6-bisphosphate + H2O Prof. Dr. Edilbert Van Driessche ATP + H2O ADP + fructose-1,6-bisphosphate FBPase-1 fructose-6-phosphate + Pi ADP + Pi + heat Requirement for a coordinated regulation of both pathways Differential regulation of hexokinase isoenzymes in liver and muscle by glucose-6-phosphate Four hexokinase isoenzymes exist, encoded by 4 different genes (isoenzymes are different proteins that catalyze the same reaction) In myocytes: predominantly hexokinase II (also I and III) In hepathocytes: predominantly hexokinase IV (also called glucokinase) Prof. Dr. Edilbert Van Driessche The different hexokinase isoenzymes of liver and muscle reflect the different roles of these organs in carbohydrate metabolism. Muscle: consumption of glucose for the production of energy Liver: maintains blood glucose homeostasis by removing or producing glucose depending on the prevailing glucose concentration Comparison between hexokinase II and hexokinase IV Prof. Dr. Edilbert Van Driessche Hexokinase I: very high affinity for glucose (half saturated at 0.1 mM). At intracellular [glucose] it acts at or near its maximal rate. In muscle, hexokinase I & II are allosterically inhibited by glucose-6P. In liver, hexokinase IV is half saturated at [glucose] of about 10 mM, i.e. higher than the [glucose] in the blood (4-5 mM). Hexokinase IV can respond to the level of glucose in the blood. Prof. Dr. Edilbert Van Driessche Regulation of hexokinase IV by sequestration in the nucleus After a meal, blood glucose concentration increases and excess glucose is transported into the hepatocytes and converted by hexokinase IV into glucose-6P. Because hexokinase IV is not saturated at 10 mM, its activity continues to increase as the [glucose] increases to 10 mM or more. Hexokinase IV is inhibited by the reversible binding of a regulatory protein that is specific for the liver. Hexokinase IV is NOT inhibited by glucose-6P. The complex control of phosphofructokinase I (PFK-1) Remember: Glucose-6P glycolysis glycogen synthesis pentosephosphate pathway Is a major regulatory enzyme of glycolysis !! Prof. Dr. Edilbert Van Driessche The metabolically irreversible reaction catalyzed by PFK-1 commits glucose to glycolysis. PFK-1: High levels of ATP lower the affinity for fructose-6P. ADP & AMP allosterically releave inhibition by ATP. Citrate allosterically increases the effect of ATP. Is allosterically activated by fructose-2,6-bisphosphate. The central role of citrate: Is a key intermediate in the aerobic oxidation of pyruvate, fatty acids and amino acids. Prof. Dr. Edilbert Van Driessche Regulation of pyruvate kinase All three isozymes of pyruvate kinase are allosterically inhibited by ATP, acetyl-CoA and long chain fatty acids (all signs of an abundant energy supply). The liver isoenzyme (L form), but not the muscle isoenzyme (M form) is further regulated by phosphorylation. When the glucose level in blood decreases, glucagon release causes a cyclic-AMPdependent phosphorylation of the L form, that inactivates it; use of glucose in the liver is slowed down to save the available glucose as fuel for the brain and other organs. Regulation of gluconeogenesis Remember: pyruvate glucose Fate of pyruvate: Pyruvate in the mitochondria can either be transformed to acetyl-CoA, or to oxaloacetate. Prof. Dr. Edilbert Van Driessche Q: Which way will be choosen? A: This depends mainly on the availability of acetyl-CoA. When fatty acids are used as fuel molecules, their breakdown yields acetyl-CoA, a signal that further oxidation of glucose for fuel is not required. Conclusion: When sufficient concentrations of citrate and ATP are present, gluconeogenesis is favored. I. The foundations of biochemistry 1. 2. The cell and subcellular organelles Short overview of biomolecules: proteins, nucleic acids, lipids, polysaccharides, and their building blocks Fundamentals of energy and energy transfer 3. Prof. Dr. Edilbert Van Driessche II. Metabolism and its regulation 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Strategies to regulate metabolic pathways Glycolysis and gluconeogenesis Pentose-phosphate pathway and Calvin cycle Glycogen metabolism Fatty acid degradation (-oxidation and -oxidation) Fatty acid biosynthesis and ketone bodies Pyruvate dehydrogenase, citric acid cycle and anaplerotic reactions Amino acid degradation and urea cycle Nitrogen metabolism: biosynthesis of amino acids, nucleotides and related molecules Oxidative phosphorylation and ATP synthesis Photosynthesis (light reactions) III. Intra- and intercellular organization 15. 16. 17. 18. 19. 20. Biomembranes (including transport systems) The cytoskeleton Metabolons: physical organization of metabolic pathways Protein trafficking Protein degradation Integration of metabolism: from molecules to cells to organs Pentose-phosphate pathway Prof. Dr. Edilbert Van Driessche Aims: – formation of pentose-phosphates through the oxidation of glucose-6P – generation of reducing power, i.e. NADPH for reductive biosynthetic processes Generation of pentose-phosphates End products: D-ribose 5-phosphate CO2 Prof. Dr. Edilbert Van Driessche NADPH Recycling of pentose-phosphates to glucose-6P Epimerization of ribulose 5-phosphate: Prof. Dr. Edilbert Van Driessche Non-oxidative reactions of the pentose-phosphate pathway Reactions catalyzed by transketolase and transaldolase Prof. Dr. Edilbert Van Driessche Transketolase: transfers a 2-carbon fragment from a ketose donor to an aldose acceptor. Reactions catalyzed by transketolase and transaldolase Prof. Dr. Edilbert Van Driessche Transaldolase: transfers a 3-carbon fragment from a ketose donor to an aldose acceptor. Partitioning of glucose-6P between glycolysis and pentose-phosphate pathway Prof. Dr. Edilbert Van Driessche When NADPH is formed faster than being used in biosynthetic processes, the [NADPH] rises and inhibits the first enzyme of the pentose-phosphate pathway. Glucose-6P is available for glycolysis Prof. Dr. Edilbert Van Driessche