1. Introduction to Metabolism

2. Metabolism (The Acquisition and Utilization of Free Energy) Catabolism: exergonic oxidation Anabolism: endergonic processes

3. Endergonic Processes Mechanical Work Active Transport Biosynthesis

4. Anabolism and Catabolism exergonic endergonic

5. Roles of ATP and NADP + in Metabolism

6. ATP Kinetic Stability of Phosphoanhydride Bonds

7. ATP Adenosine Ribose Triphosphate

8. Hydrolysis of ATP

9. Phosphate Compounds

10. Roles of ATP (Coupled Reactions) ∆G o’ (kJ/mol) ---------- Fructose-6-P + P i ——> Fructose-1,6-bisP + H 2 O +13.3 ATP + H 2 O ——> ADP + P i -30.5 ------------------------------------------------------------------------------- Fructose-6-P + ATP ——> Fructose-1,6-bisP + ADP -17.2

11. Roles of ATP Early stages of nutrient breakdown Glucose + ATP ——> Glucose-6-P + ADP Interconverson of nucleoside triphosphtes NDP + ATP ——> NTP + ADP Nucleoside Diphosphate Kinase Physiological processes –Muscle contraction –Active transport

12. Roles of ATP Additional phosphoanhydride cleavages in highly endergonic reactons (NMP) n + NTP ——> (NMP) n+1 + PP i PP i + H 2 O ——> 2 P i Pyrophosphatase

13. Sources of ATP Phototrophs: photosynthesis Chemotrophs: oxidation of organic compounds (e.g. carbohydrates, lipids, and proteins)

14. Formation of ATP Adenylate Kinase reaction 2 ADP ——> AMP + ATP Substrate-level phosphorylation X–P + ADP ——> X–H + ATP Oxidative phosphorylation Photophosphorylation

15. Substrate-Level Phosphorylation

16. Oxidative Phosphorylation

17. Photophosphorylation

18. Source of NAD(P)+, and other cofactors

20. NADP + Nicotinamide Adenine Dinucleotide (Phosphate)

21. Figure 14-1 Niacin

22. Figure 14-11 Reduction of NAD + or NADP + to NADH or NADPH

23. Metabolic Pathways A ——> B ——> C ——> D ——> E Metabolites Enzymes

24. Metabolic Map

26. Figure 14-3 Overview of Catabolism

27. Properties of Metabolic Pathways Separate Anabolic and Catabolic Pathways Steady-State Irreversible (overall): reversibility of individual steps First Committed (Exergonic) Step: others close to equilibrium Compartmentation (organelles & tissues): isoenzymes and transport Regulation (usually first committed step): often rate-limiting

28. Potential Futile Cycles (Regulation)

29. Steady State

30. Thermodynamics of individual steps A  B  G o’ = -RTlnK eq Not standard conditions or at equilibrium:  G =  G o’ +RTln([B]/[A]) Three Physiological Conditions:  G o’ <<<<<<0 :  G always negative Example: ATP hydrolysis  G o’ >0 : near equilibrium, reversible, direction depends on actual [B]/[A] Example: Most reactions  G o’ >>>>>>0 :  G always positive, must be coupled Example: Phosphorylation of Glucose

31.  G o’ >0

33. Regulation of Metabolic Pathways Specific Controls General Controls

34. Specific Controls Control of Enzyme Amount –Constitutive Enzymes –Inducible Enzymes –Repressible Enzymes Control of Enzyme Activity –Regulatory Enzymes –Effectors (Ligands)

35. General Controls (Integration of Cellular or Organism Functions) Internal Effectors –Catabolite Repression –Energy Charge –Reduction Potential External Effectors (e.g. hormones) Significance: Efficiency and Flexibility!

36. Types of Reactions

37. Group Transfer Reactions

38. Phosphoryl Group Transfer

39. Elimination Reactions

40. Isomerization Reactions (Intramolecular Hydrogen Shifts)

41. Making C-C Bonds Note: thioester

42. Breaking C-C Bonds

43. Oxidation-Reduction Reactions SH 2 + NAD + + H 2 O ——> S + NADH + H 3 O + SH 2 : Reduced Substrate S: Oxidized Product NAD + : Electron Acceptor FAD: Electron Acceptor

44. Figure 14-11 Reduction of NAD + to NADH

45. Figure 14-12 Flavin Adenine Dinucleotide (FAD)

46. Figure 14-13 part 1 Reduction of FAD to FADH 2

47. Figure 14-13 part 2 Reduction of FAD to FADH 2

48. One Electron Oxidation-Reduction Reactions

49. Half-Reactions Oxidation Involves(e - of H: - ) Loss Reduction Involves(e - of H: - ) Gain

50. Alcohol Dehydrogenase (Oxidation-Reduction Reaction)

51. Experimental Approaches to Metabolism

52. Features of Metabolic Pathways A ——> B ——> C ——> D ——> E (1)Sequences and Energetics (2) Enzymes and Mechanisms (3) Control Mechanisms (Regulation) (4) Compartmentation

53. Elucidation of Metabolic Pathways A ——> B ——> C ——> D ——> E Metabolic Inhibitors: accumulation of intermediates Biochemical Genetics: mutants Pathway Labeling: isotopes

54. Metabolic Inhibitors (Accumulation of Intermediates) (e.g. Glycolysis) Fluoride: (2-phosphoglycerate and consequently 3–phosphoglycerate)

55. Biochemical Genetics (Mutants) Natural Genetic Defects Manipulation of Microorganisms Accumulation of Intermediates Growth Requirements (auxotrophic mutants) A ——> B ——> C ——> D ——> E

56. Pathway Labeling A* ——> B* ——> C* Stable Isotopes Radioisotopes

57. Detection of Isotopes Stable Isotopes –Mass Spectrometry –NMR Radioisotopes –Proportional Counting (Geiger Counter) –Liquid Scintillation Counting –Autoradiography

58. Quantify Differential Expression Condition 1Condition 2 Sample Prep Mix samples and detect Quantify Differences

59. Control of Expression Transcription: –Microarray Proteomics –2D-SDS-PAGE –Isotope Coded Affinity Tag

60. ICAT Chemistry IAM Biotin Reactive Group (specific for cysteines) Affinity Tag Isotope code (D or 13 C) LIGHT HEAVY ICAT = Isotope Coded Affinity Tag Same behavior chemically, but different in mass.