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General Biochemistry Part II-d Prof. Dr. S. Beeckmans Prof. Dr. E. Van Driessche [email protected] Protein Chemistry Lab Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, room E.5.23 Pictures are taken from: Lehninger PRINCIPLES OF BIOCHEMISTRY (4th Edition) − 2005 by: David L. Nelson & Michael M. Cox W.H. Freeman and Comp., New York BIOCHEMISTRY (5th Edition) − 2002 by: Jeremy M Berg, John L Tymoczko & Lubert Stryer W.H. Freeman and Comp., New York BIOCHEMISTRY Prof. Dr. Sonia Beeckmans (3rd Edition) − 2003 by: Donald Voet & Judith G Voet John Wiley and Sons 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. II. Metabolism and its regulation Prof. Dr. Sonia Beeckmans 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 (β-oxidation and ω-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 Overview of metabolism Proteins Fats Saccharides Prof. Dr. Sonia Beeckmans FOOD Prof. Dr. Sonia Beeckmans Acetyl-CoA is an energy-rich molecule 1 kcal = 4.18 kJ Prof. Dr. Sonia Beeckmans ATP Prof. Dr. Sonia Beeckmans Mitochondria The conversion of pyruvate to acetyl-CoA is catalyzed by a complex Cryoelectron micrograph of bovine PDHC. Prof. Dr. Sonia Beeckmans Samples are viewed at extremely low temperatures, thus avoiding potential artefacts introduced by the usual process of dehydrating + fixing + staining. Prof. Dr. Sonia Beeckmans The pyruvate dehydrogenase complex consists of 3 enzymes that cooperate and form a multienzyme complex The pyruvate dehydrogenase complex requires five coenzymes Prof. Dr. Sonia Beeckmans 1.5 nm swinging arm Prof. Dr. Sonia Beeckmans The pyruvate dehydrogenase complex requires five coenzymes Prof. Dr. Sonia Beeckmans The pyruvate dehydrogenase complex requires five coenzymes 3D model of bovine PDHC E1 (pyruvate dehydrogenase) is at the outside. The core consists of 60 molecules E2 (dihydrolipoyl transacetylase). The lipoyl domain of E2 reaches outward touching the active sites of molecules E1. Prof. Dr. Sonia Beeckmans E3 (dihydrolipoyl dehydrogenase) is also supposed to be bound to the core. E2 consists of three types of domains connected by short linkers: Consecutive reactions of PDHC + TPP HETPP + CO2 E1 HETPP + lipoamide Prof. Dr. Sonia Beeckmans E2 TPP + Acetyl-lipoamide Acetyl-lipoamide + CoA-SH Acetyl-CoA + Dihydrolipoamide Dihydrolipoamide + FAD Lipoamide + FADH2 NAD+ E3 NADH + H+ PROBLEM !!! For energetic reasons this reaction is not possible: FAD FADH2 NAD+ NADH + H+ Solution of this problem: FAD E3 S Prof. Dr. Sonia Beeckmans S FAD°H S° NADH + H+ NAD+ SH Prof. Dr. Sonia Beeckmans —S-S— • —S —SH x !!! Prof. Dr. Sonia Beeckmans Generation of Acetyl-CoA ?? Prof. Dr. Sonia Beeckmans Chapter: The citric acid cycle Prof. Dr. Sonia Beeckmans When you study this reaction pathway, try to remember the formulas of the intermediates, not their names !!!!! Preview The citric acid cycle Stoichiometry: Acetyl-CoA + 3NAD+ + FAD + ADP + Pi + 2H2O 2CO2 + 3(NADH + H+) + FADH2 + ATP + CoASH 2CO2 leaving are different from those entering 4 pairs of H atoms leave the cycle Prof. Dr. Sonia Beeckmans 1 high-energy phosphate bond is formed NADH and FADH2 will be reoxidized in the next pathway (OP), and ATP will be generated as electrons are transferred from these carriers to O2, the ultimate electron acceptor the cycle operates as a real cycle only under aerobic conditions, i.e. when OP pathway occurs Action of citrate synthase Prof. Dr. Sonia Beeckmans Citrate synthase is the driving force for the citric acid cycle (this is why an energy-rich acetyl-CoA molecule is consumed, instead of performing the reaction: CH3-COOH + OAA citrate). Prof. Dr. Sonia Beeckmans Action of citrate synthase Action of aconitase Prof. Dr. Sonia Beeckmans Substrate binding involves a “non-haem iron sulfur cluster”. Action of isocitrate dehydrogenase Mn2+ in the enzyme’s active site interacts with the carbonyl group of the intermediate oxalosuccinate. Prof. Dr. Sonia Beeckmans It also stabilizes the enol formed transiently by decarboxylation. There are two different ICDHases, one acting with NAD (found in mitochondria) and another one acting with NADP (both in mito and in cytoplasm). The function of the latter may be generation of NADPH for anabolism. Prof. Dr. Sonia Beeckmans Action of α-ketoglutarate dehydrogenase A complex built up of E1, E2 and E3, just like the pyruvate dehydrogenase complex. Enzyme E3 is even shared by both multi-enzyme complexes. Action of succinyl-CoA synthetase ATP ADP Nucleoside diphosphate kinase Coenzyme A Prof. Dr. Sonia Beeckmans phosphate The enzyme is built up of 2 different subunits. The active site includes part of both the α- (blue) and the β-subunit (brown). Prof. Dr. Sonia Beeckmans Action of succinyl-CoA synthetase Such process is called “substrate-level phosphorylation”. Prof. Dr. Sonia Beeckmans Action of succinate dehydrogenase SDH is an inner membrane enzyme. All other CAC enzymes are in the mitochondrial matrix. Prof. Dr. Sonia Beeckmans Action of fumarase Fumarase is highly stereospecific: maleate is not a substrate and neither is D-malate. Action of malate dehydrogenase ΔG = –2.303*RT*logKeq Keq = 0.00000618 Prof. Dr. Sonia Beeckmans = [OAA] [NADH] [malate] [NAD+] The equilibrium of this reaction lies far to the left under standard conditions. In cells, the concentration of OAA is extremely low (<10–6 M), pulling the malate dehydrogenase reaction towards the formation of OAA. Prof. Dr. Sonia Beeckmans Citrate: a symmetrical molecule that reacts asymmetrically Prof. Dr. Sonia Beeckmans Enzymes of the citric acid cycle Anaerobic bacteria have an incomplete cycle They miss the α-ketoglutarate dehydrogenase complex. Prof. Dr. Sonia Beeckmans The lower branch now runs in opposite direction, and will be pushed forward by the ΔG = –7.1 kcal/mol of malate dehydrogenase. The citric acid cycle is called an amphibolic pathway (serves 2 different purposes): – source of energy – source of intermediates for anabolism. Regulation of PDHC and citric acid cycle Only in vitro, using free ATP !!! Prof. Dr. Sonia Beeckmans But: inside cells, ATP and ADP are chelated Regulation of PDHC and citric acid cycle acetyl-CoA NADH CO2 Prof. Dr. Sonia Beeckmans NAD+ pyruvate coenzyme A Role of citric acid cycle in anabolism Prof. Dr. Sonia Beeckmans Gluconeogenesis Fatty acid biosynthesis Biosynthesis of purines/pyrimidines Biosynthesis of amino acids In order to remove intermediates, intermediates need to be replenished Prof. Dr. Sonia Beeckmans Role of citric acid cycle in anabolism ANAPLEROTIC REACTIONS Pyruvate carboxylase ATP HCO3– pyruvate Prof. Dr. Sonia Beeckmans Acetyl-CoA An allosteric activator prevents waste of ATP: the enzyme is inactive in the absence of acetyl-CoA. A long flexible arm allows the prosthetic group to rotate from site 1 to site 2. A high-energy substrate Other anaplerotic pathways: Prof. Dr. Sonia Beeckmans Glyoxylic acid cycle Amino acid degradation The glyoxylic acid cycle Allows the synthesis of sugars exclusively from fatty acids. Occurs in bacteria/yeasts, when they grow on e.g. ethanol, ... Prof. Dr. Sonia Beeckmans Occurs in some plant seedlings. Citric acid cycle Prof. Dr. Sonia Beeckmans In plant seedlings In bacteria Prof. Dr. Sonia Beeckmans Coordinated regulation of glyoxylic acid cycle and citric acid cycle occurs through phosphorylation of ICDH. A high-energy substrate Other anaplerotic pathways: Prof. Dr. Sonia Beeckmans Glyoxylic acid cycle Amino acid degradation