1. Biochemistry 432/832 February 21 Chapters 27 and 28 Nucleic acid metabolism Integration of metabolic pathways

2. Announcements: -

3. DNA synthesis Synthesis of deoxyribo- nucleotides --- reduction at the 2’-position of the ribose ring of nucleoside diphosphates

4. Deoxyribonucleotide Biosynthesis Reduction at 2’-position commits nucleotides to DNA synthesis Replacement of 2’-OH with hydride is catalyzed by ribonucleotide reductase An  2  2 -type enzyme - subunits R 1 (86 kDa) and R 2 (43.5 kDa) R 1 has two regulatory sites, a specificity site and an overall activity site

5. E.coli ribonucleotide reductase

6. Regulation of deoxynucleotide synthesis

7. Synthesis of dTMP

8. Synthesis of Thymine Nucleotides Thymine nucleotides are made from dUMP, which derives from dUDP, dCDP dUDP  dUTP  dUMP  dTMP dCDP  dCMP  dUMP  dTMP Thymidylate synthase methylates dUMP at 5-position to make dTMP N 5,N 10 -methylene THF is 1-C donor Role of 5-FU in chemotherapy

9. The dCMP deaminase reaction

10. The thymidylate synthase reaction

11. Structure of fluoro compounds - thymine analogs - inhibitors of DNA synthesis

12. Integration of metabolic pathways

13. 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 - sugar- phosphates, alpha-keto acids, CoA derivatives, and PEP ATP & NADPH couple catabolism & anabolism Phototrophs also have photosynthesis and CO 2 fixation systems

14. Intermediary metabolism

15. 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

16. 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

17. 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

18. 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!

19. Nature of the ATP Equivalent A different perspective  G for ATP hydrolysis says that at equilibrium the concentrations of ADP and P i 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][P i ] ratio stays very high This allows ATP hydrolysis to serve as the driving force for nearly all biochemical processes

20. 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!

21. Substrate cycles

22. 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

23. ATP coupling coefficients for fatty acid oxidation and synthesis

24. ‘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

25. Relative concentrations of AMP, ADP and ATP as a function of energy charge

26. Responses of regulatory enzymes to variation in energy charge Catabolic Anabolic

27. The oscillation of energy charge as a consequence of R and U processes

28. 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

29. 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 O 2 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! Metabolism in a multicellular organism

30. Muscle Muscles must be prepared for rapid provision of energy Resting state: 30% of O 2, exercise: 90% of O 2 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)

31. 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

32. Adipose tissue Function: storage depot for fatty acids release of f.a. into bloodstream

33. 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)

34. 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)

35. Metabolic conversion of glucose-6- phosphate in the liver Common intermediates

36. Analyses of individual enzymes of pathways Inhibitor analyses, radioisotopes, compartmentalization Parallel analyses of thousands of enzymes or pathways Bioinformatics, functional genomics, expression analyses, proteomics Methods to study metabolism