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Biochemistry 432/832 February 21 Chapters 27 and 28 Nucleic acid metabolism Integration of metabolic pathways Announcements: - DNA synthesis Synthesis of deoxyribonucleotides --reduction at the 2’-position of the ribose ring of nucleoside diphosphates Deoxyribonucleotide Biosynthesis • Reduction at 2’-position commits nucleotides to DNA synthesis • Replacement of 2’-OH with hydride is catalyzed by ribonucleotide reductase • An 22-type enzyme - subunits R1 (86 kDa) and R2 (43.5 kDa) • R1 has two regulatory sites, a specificity site and an overall activity site E.coli ribonucleotide reductase Regulation of deoxynucleotide synthesis Synthesis of dTMP Synthesis of Thymine Nucleotides • Thymine nucleotides are made from dUMP, which derives from dUDP, dCDP • dUDPdUTPdUMPdTMP • dCDPdCMPdUMPdTMP • Thymidylate synthase methylates dUMP at 5-position to make dTMP • N5,N10-methylene THF is 1-C donor • Role of 5-FU in chemotherapy The dCMP deaminase reaction The thymidylate synthase reaction Structure of fluoro compounds - thymine analogs inhibitors of DNA synthesis Integration of metabolic pathways Systems Analysis of Metabolism • • • • Catabolic and anabolic pathways, occurring simultaneously, must act as a regulated, orderly, responsive whole catabolism, anabolism and macromolecular synthesis Just a few intermediates connect major systems - sugarphosphates, alpha-keto acids, CoA derivatives, and PEP ATP & NADPH couple catabolism & anabolism Phototrophs also have photosynthesis and CO2 fixation systems Intermediary metabolism 28.2 Metabolic Stoichiometry Three types of stoichiometry in biological systems • Reaction stoichiometry - the number of each kind of atom in a reaction • Obligate coupling stoichiometry - the required coupling of electron carriers • Evolved coupling stoichiometry - the number of ATP molecules that pathways have evolved to consume or produce The Significance of 38 ATPs • Glucose oxidation • If 38 ATP are produced, cellular G is -967 kJ/mol • If G = 0, 58 ATP could be made • So the number of 38 is a compromise The ATP Equivalent What is the "coupling coefficient" for ATP produced or consumed? • Coupling coefficient is the moles of ATP produced or consumed per mole of substrate converted (or product formed) • Cellular oxidation of glucose has a coupling coefficient of 30-38 (depending on cell type) • Hexokinase has a coupling coefficient of -1 • Pyruvate kinase (in glycolysis) has a coupling coefficient of +1 The ATP Value of NADH vs NADPH • The ATP value of NADH is 2.5-3 • The ATP value of NADPH is higher • NADPH carries electrons from catabolic pathways to biosynthetic processes • [NADPH]>[NADP+] so NADPH/NADP+ is a better e- donating system than NADH/NAD • So NADPH is worth 3.5-4 ATP! Nature of the ATP Equivalent • • • A different perspective G for ATP hydrolysis says that at equilibrium the concentrations of ADP and Pi should be vastly greater than that of ATP However, a cell where this is true is dead Kinetic controls over catabolic pathways ensure that the [ATP]/[ADP][Pi] ratio stays very high This allows ATP hydrolysis to serve as the driving force for nearly all biochemical processes Substrate Cycles If ATP c.c. for a reaction in one direction differs from c.c. in the other, the reactions can form a substrate cycle • The point is not that ATP can be consumed by cycling • But rather that the difference in c.c. permits both reactions (pathways) to be thermodynamically favorable at all times • Allosteric effectors can thus choose the direction and/or regulate flux in the pathway! Substrate cycles Unidirectionality of Pathways • • • • A "secret" role of ATP in metabolism Metabolic pathways proceed in one direction Either catabolic or anabolic, not both Both directions of any pair of opposing pathways must be favorable, so that allosteric effectors can control the direction effectively The ATP coupling coefficient for any such sequence has evolved so that the overall equilibrium for the conversion is highly favorable ATP coupling coefficients for fatty acid oxidation and synthesis ‘Energy Charge’ • Adenylates provide phosphoryl groups to drive thermodynamically unfavorable reactions • Energy charge is an index of how fully charged adenylates are with phosphoric anhydrides (number of phosphoric anhydrate bonds divided by total adenylate pool) • E.C. = (2ATP+ADP) / 2 (ATP+ADP+AMP) • If [ATP] is high, E.C.1.0 • If [ATP] is low, E.C. 0 Relative concentrations of AMP, ADP and ATP as a function of energy charge Responses of regulatory enzymes to variation in energy charge Catabolic Anabolic The oscillation of energy charge as a consequence of R and U processes Metabolism in a multicellular organism • Organs and tissues have metabolic profiles (specialized) • Reflect metabolic function • Brain - glucose uptake • Muscle - Cori cycle (lactate) • Adipose - storage of fat • Liver - glucose synthesis • Heart - prefers fatty acids as fuel (no storage) • Differences: function, preferred fuel, whether or not fuel stored, what energy precursors they exploit Metabolism in a multicellular organism Fueling the Brain • Brain has very high metabolism but has no fuel reserves • This means brain needs a constant supply of glucose • 120 g glucose and 20% of O2 consumes, mass of brain is 2% • In fasting conditions, brain can use ketone bodies (from fatty acids) • This allows brain to use fat as fuel! Muscle • Muscles must be prepared for rapid provision of energy • Resting state: 30% of O2, exercise: 90% of O2 • Fuel source: glucose (exercise), fatty acids (resting state) • Stored fuel: Glycogen (local) provides additional energy, releasing glucose for glycolysis • No export of glucose (lactate is exported) Muscle Protein Degradation • During fasting or high activity, amino acids are degraded to pyruvate, which can be transaminated to alanine • Alanine circulates to liver, where it is converted back to pyruvate - food for gluconeogenesis • This is a fuel of last resort for the fasting or exhausted organism Adipose tissue • Function: storage depot for fatty acids • release of f.a. into bloodstream Liver • Function: main metabolic processing center • Regulates glucose metabolism (blood G <-> liver G <-> glycogen) • Regulates fat metabolism • Fed conditions (synthesis of f.a. ->TAG -> storage) • Fasting (srorage ->f.a.-> acetyl-CoA) Control of metabolic pathways • Substrate/product activation/inhibition (product of a pathway inhibits committed step; substrate activates the pathway) • allosteric control (binding of an effector at one site affects enzyme activity at another site) • covalent control (phosphorylation, adenylylation, redox, etc) • gene expression – requires time (transcription - RNA synthesis, translation - protein synthesis) Common intermediates Metabolic conversion of glucose-6phosphate in the liver Methods to study metabolism Analyses of individual enzymes of pathways Inhibitor analyses, radioisotopes, compartmentalization Parallel analyses of thousands of enzymes or pathways Bioinformatics, functional genomics, expression analyses, proteomics