1. Introduction to Metabolism A hummingbird has a rapid rate of metabolism, but its basic metabolic reactions are the same as those in many diverse organisms

2. Prentice Hall c2002Chapter 102 Autotrophs – use CO 2 as sole carbon source (plants, photosynthetic bacteria, etc.) Heterotrophs-obtain carbon from their environment Constant cycling of material between autotrophs and heterotrophs

3. Prentice Hall c2002Chapter 103

4. Metabolism Is the Sum of Cellular Reactions Metabolism - the entire network of chemical reactions carried out by living cells Metabolites - small molecule intermediates in the degradation and synthesis of polymers Catabolic reactions - degrade molecules to create smaller molecules and energy Anabolic reactions - synthesize molecules for cell maintenance, growth and reproduction

5. Anabolism and catabolism

6. Prentice Hall c2002Chapter 106

7. Metabolic Reactions Metabolism includes all enzyme catalyzed reactions The metabolism of the four major groups of biomolecules will be considered: Carbohydrates Lipids Amino Acids Nucleotides

8. Prentice Hall c2002Chapter 108 Organization of Metabolic Reactions Occur via pathways – series of organized reaction steps Compartmentalized – certain reactions occur in particular cells, organelles or other specific sites Pathways are regulated – controlled –to keep anabolism and catabolism reactions separate (some use the same enzymes) –Timing to produce products only when necessary –At least one step in a pathway needs to be irreversible (exergonic, -  G)

9. Prentice Hall c2002Chapter 109 Types of pathways Individual reaction series –Linear (can branch out) –Cyclic –Spiral Connecting pathways –Converging (metabolic) –Diverging (anabolic)

10. Forms of metabolic pathways (a)Linear (b) Cyclic or branched

11. (c) Spiral pathway (fatty acid biosynthesis)

12. Prentice Hall c2002Chapter 1012

13. Metabolism Proceeds by Discrete Steps Multiple-step pathways permit control of energy input and output Catabolic multi-step pathways provide energy in smaller stepwise amounts) Each enzyme in a multi-step pathway usually catalyzes only one single step in the pathway Control points occur in multistep pathways

14. Single-step vs multi- step pathways A multistep enzyme pathway releases energy in smaller amounts that can be used by the cell

15. Metabolic Pathways Are Regulated Regulation permits response to changing conditions Common ways to regulate (1) Supply of substrates(concentration) (2) Removal of products (3) Pathway enzyme activities Allosteric regulation Covalent modification

16. Feedback inhibition Product of a pathway controls the rate of its own synthesis by inhibiting an early step (usually the first “committed” step (unique to the pathway)

17. Feed-forward activation Metabolite early in the pathway activates an enzyme further down the pathway

18. Covalent modification for enzyme regulation Interconvertible enzyme activity can be rapidly and reversibly altered by covalent modification Protein kinases phosphorylate enzymes (+ ATP) Protein phosphatases remove phosphoryl groups The initial signal may be amplified by the “cascade” nature of this signaling

19. Prentice Hall c2002Chapter 1019 Reaction Types in Pathways 1.Oxidation-Reduction (Redox) 2.Making or breaking C-C bonds 3.Internal rearrangements, isomerizations or eliminations 4.Group transfers 5.Free radical reactions

20. Prentice Hall c2002Chapter 1020 Redox reactions Oxidation – loss of electrons, gain of oxygen, loss of hydrogen –Hydrogenases –Oxidases Note the different oxidation states of carbon

21. Prentice Hall c2002Chapter 1021

22. Prentice Hall c2002Chapter 1022

23. Prentice Hall c2002Chapter 1023 Carbon-Carbon Bonds Bond cleavage –Homolytic (1 electron for each atom) –Heterolytic (both electrons to one atom) –Recall Nucleophiles (attracted to + charges) Electrophiles (attracted to – charges)

24. Prentice Hall c2002Chapter 1024

25. Prentice Hall c2002Chapter 1025 Common Reaction Types Many use the carbonyl group C=O  + on Carbon;  - on Oxygen –Reactive group in Aldol condensations Claisen condensations Decarboxylations

26. Prentice Hall c2002Chapter 1026

27. Prentice Hall c2002Chapter 1027 Internal Reactions Rearrangements, isomerizations, eliminations –Groups –Bonds –Atoms

28. Prentice Hall c2002Chapter 1028

29. Prentice Hall c2002Chapter 1029

30. Prentice Hall c2002Chapter 1030 Group Transfers There are many groups to transfer –Acyl –Glycosyl –Phosphoryl Phosphate = P i Pyrophosphate = PP i

31. Prentice Hall c2002Chapter 1031

32. Prentice Hall c2002Chapter 1032 Free Radicals Unpaired electrons More common than previously thought

33. 10.3 Major Pathways in Cells Metabolic fuels Three major nutrients consumed by mammals: (1) Carbohydrates - provide energy (2) Proteins - provide amino acids for protein synthesis and some energy (3) Fats - triacylglycerols provide energy and also lipids for membrane synthesis

34. Fig 10.5 Overview of catabolic pathways

35. Catabolism produces compounds for energy utilization Three types of compounds are produced that mediate the release of energy (1) Acetyl CoA (2) Nucleoside triphosphates (e.g. ATP) (3) Reduced coenzymes (NADH, FADH 2, QH 2 )

36. Reducing Power Electrons of reduced coenzymes flow toward O 2 This produces a proton flow and a transmembrane potential Oxidative phosphorylation is the process by which the potential is coupled to the reaction: ADP + P i ATP

37. 10.4 Compartmentation and Interorgan Metabolism Compartmentation of metabolic processes permits: - separate pools of metabolites within a cell - simultaneous operation of opposing metabolic paths - high local concentrations of metabolites - coordinated regulation of enzymes Example: fatty acid synthesis enzymes (cytosol), fatty acid breakdown enzymes (mitochondria)

38. Fig. 10.6 Compartmentation of metabolic processes

39. 10.5 Thermodynamics and Metabolism Free-energy change (  G) is a measure of the chemical energy available from a reaction  G = G products - G reactants  H = change in enthalpy  S = change in entropy A. Free-Energy Change

40. Both entropy and enthalpy contribute to  G  G =  H - T  S (T = degrees Kelvin) -  G = a spontaneous reaction in the direction written +  G = the reaction is not spontaneous  G = 0 the reaction is at equilibrium Relationship between energy and entropy

41. The Standard State (  G o ) Conditions Reaction free-energy depends upon conditions Standard state (  G o ) - defined reference conditions Standard Temperature = 298K (25 o C) Standard Pressure = 1 atmosphere Standard Solute Concentration = 1.0M Biological standard state =  G o’ Standard H + concentration = 10 -7 (pH = 7.0) rather than 1.0M (pH = 1.0)

42. B. Equilibrium Constants and Standard Free-Energy Change For the reaction: A + BC + D  G reaction =  G o’ reaction + RT ln([C][D]/[A][B]) At equilibrium: Keq = [C][D]/[A][B] and  G reaction = 0, so that:  G o’ reaction = -RT ln K eq

43. C. Actual Free-Energy Change Determines Spontaneity of Cellular Reactions When a reaction is not at equilibrium, the actual free energy change (  G) depends upon the ratio of products to substrates Q = the mass action ratio  G =  G o’ + RT ln Q Where Q = [C]’[D]’ / [A]’[B]’

44. 10.6 The Free Energy of ATP Energy from oxidation of metabolic fuels is largely recovered in the form of ATP

45. Table 10.1

46. Fig 10.7 Hydrolysis of ATP

47. Fig 10.8 Complexes between ATP and Mg 2+

48. ATP is an “energy-rich” compound A large amount of energy is released in the hydrolysis of the phosphoanhydride bonds of ATP (and UTP, GTP, CTP) All nucleoside phosphates have nearly equal standard free energies of hydrolysis

49. Energy of phosphoanhydrides (1) Electrostatic repulsion among negatively charged oxygens of phosphoanhydrides of ATP (2) Solvation of products (ADP and P i ) or (AMP and PP i ) is better than solvation of reactant ATP (3) Products are more stable than reactants There are more delocalized electrons on ADP, P i or AMP, PP i than on ATP

50. 10.7 The Metabolic Roles of ATP Energy-rich compounds can drive biosynthetic reactions Reactions can be linked by a common energized intermediate (B-X) below A-X + BA + B-X B-X + C B + C-X

51. Glutamine synthesis requires ATP energy

52. A. Phosphoryl-Group Transfer Phosphoryl-group-transfer potential - the ability of a compound to transfer its phosphoryl group Energy-rich or high-energy compounds have group transfer potentials equal to or greater than that of ATP Low-energy compounds have group transfer potentials less than that of ATP

53. Table 10.3

54. B. Production of ATP by Phosphoryl-Group Transfer Metabolites with high phosphoryl-group-transfer potentials can donate a phosphoryl group to ADP to form ATP Energy-rich compounds are intermediates in catabolic pathways Energy storage compounds can be energy-rich

55. Fig 10.9 Relative phosphoryl-group- transfer potentials

56. Fig 10.10 Transfer of the phosphoryl group from PEP to ADP Phosphoenolpyruvate (PEP) (a glycolytic intermediate) has a high P-group transfer potential PEP can donate a P to ADP to form ATP

57. Phosphagens: Energy-rich storage molecules in animal muscle Phosphocreatine (PC) and phosphoarginine (PA) are phosphoamides Have higher group-transfer potentials than ATP Produced in muscle during times of ample ATP Used to replenish ATP when needed via creatine kinase reaction

58. Fig 10.11 Structures of PC and PA

59. C. Nucleotidyl-Group Transfer Transfer of the nucleotidyl group from ATP is another common group-transfer reaction Synthesis of acetyl CoA requires transfer of an AMP moiety to acetate Hydrolysis of pyrophosphate (PP i ) product drives reaction to completion

60. Fig 10.12 Synthesis of acetyl CoA (continued next slide)

61. Fig. 10.12 (continued)

62. 10.8 Thioesters Have High Free Energies of Hydrolysis Thioesters are energy-rich compounds (10.22) Acetyl CoA has a  G o’ = -31 kJ mol -1 (10.23)

63. Succinyl CoA Energy Can Produce GTP

64. 10.9 Reduced Coenzymes Conserve Energy from Biological Oxidations Amino acids, monosaccharides and lipids are oxidized in the catabolic pathways Oxidizing agent - accepts electrons, is reduced Reducing agent - loses electrons, is oxidized Oxidation of one molecule must be coupled with the reduction of another molecule A red + B ox A ox + B red

65. A. Free-Energy Change Is Related to Reduction Potential The reduction potential of a reducing agent is a measure of its thermodynamic reactivity The electromotive force is the measured potential difference between two half-cells Reference half-cell reaction is for hydrogen: 2H + + 2e - H 2

66. Fig 10.13 Diagram of an electrochemical cell Electrons flow through external circuit from Zn electrode to the Cu electrode

68. Standard reduction potentials and free energy  G o’ = -nF  E o’ Relationship between standard free-energy change and the standard reduction potential: n = # electrons transferred F = Faraday constant (96.48 kJ V -1 )  E o’ = E o’ electron acceptor - E o’ electron donor

69. Actual reduction potentials (  E) Under biological conditions, reactants are not present at standard concentrations of 1 M Actual reduction potential (  E) is dependent upon the concentrations of reactants and products  E =  E o’ - (RT/nF) ln ([A ox ][B red ] / [A red ][B ox ] )

70. B. Electron Transfer from NADH Provides Free Energy Most NADH formed in metabolic reactions in aerobic cells is oxidized by the respiratory electron-transport chain Energy used to produce ATP from ADP, P i Half-reaction for overall oxidation of NADH: NAD + + 2H + + 2e - NADH + H + (E o’ = -0.32V)

71. 10.10 Experimental Methods for Studying Metabolism Add labeled substrate to tissues, cells, and follow emergence of intermediates Use sensitive isotopic tracers ( 3 H, 14 C etc) Verify pathway steps in vitro by using isolated enzymes and substrates Use metabolic inhibitors to identify individual steps and sequence of enzymes in a pathway