陳泰源 博士 中央研究院 生物化學研究所 2007/4/24 National Taiwan Normal University

2. 陳泰源 博士 中央研究院 生物化學研究所 2007/4/24 National Taiwan Normal University

3. Organisms can be classified according to their source of energy (sunlight or oxidizable chemical compounds) and their source of carbon for the synthesis of cellular material.

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

7. 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

8. Overview of catabolic pathways

9. Nucleophiles: functional groups rich in electrons and capable of donating them Electrophiles: electron-deficient functional groups that seek electrons The relative electronegativities: F>O>N>C=S>P=H

10. Cleavage of a C-C or C-H bond

11. Carbon-carbon bond formation reactions

12. Oxidation-reduction reactions

14. 13.1Bioenergetics and Thermodynamics

15. Biological Energy transformations obey the Laws of Thermodynamics • For any physical or chemical change, the total amount of energy in the universe remains constant; but it cannot be created or destroyed. • The universe always tends toward increasing disorder: in all natural processes, the entropy of the universe increases. • Living cells and organisms are open system, exchanging both material and energy with their surroundings; • living systems are never at equilibrium with their surrounding, and the constant transactions between system and surrounding explain how organisms can create order within themselves while operating within the second law of the thermodynamics.

16. 13.1Bioenergetics and Thermodynamics A. Free-Energy Change • Free-energy change(DG) is a measure of the chemicalenergyavailablefromareaction • DG = Gproducts - Greactants • DH = change in enthalpy • DS = change in entropy

17. Both entropy and enthalpy contribute to DG • DG = DH - TDS • (T = degrees Kelvin) • -DG = a spontaneous reaction in the direction written • +DG= the reaction is not spontaneous • DG= 0 the reaction is at equilibrium Relationship between energy and entropy

18. Standard Free-Energy Change(DGo) • Reaction free-energy depends upon conditions • Standard state(DGo)- defined reference conditions • Standard Temperature = 298K (25oC) • Standard Pressure = 1 atmosphere • Standard Solute Concentration = 1.0M • Standard transformed constant =DGo’ • Standard H+ concentration = 10-7 (pH = 7.0) • H2O concentration = 55.5 M • Mg2+ concentration = 1 mM

19. For the reaction: aA + bB cC + dD Equilibrium Constants and Standard Free-Energy Change DGreaction = DGo’reaction + RT ln([C]c[D]d/[A]a[B]b) • At equilibrium: Keq = [C][D]/[A][B] and DGreaction = 0, so that: DGo’reaction = -RT ln Keq

20. The standard free-energy change is directly related to the equilibrium constant

21. Energy coupling in mechanical and chemical processes. (a) The downward motion of an object releases potential energy that can do mechanical work. The potential energy made available by spontaneous downward motion, an exergonic process (pink), can be coupled to the endergonic upward movement of another object (blue). (b) In reaction 1, the formation of glucose 6-phosphate from glucose and inorganic phosphate (Pi) yields a product of higher energy than the two reactants. For this endergonic reaction, △G is positive. In reaction 2, the exergonic breakdown of adenosine triphosphate (ATP) can drive an endergonic reaction when the two reactions are coupled. The exergonic reaction has a large, negative free-energy change (△G2), and the endergonic reaction has a smaller, positive free-energy change (△G1). The third reaction accomplishes the sum of reactions 1 and 2, and the free-energy change, △G3, is the arithmetic sum of _G1 and △G2. Because △G3 is negative, the overall reaction is exergonic and proceeds spontaneously.

22. Standard free-energy changes are additive

23. Equilibrium constants are multiplicative

24. 13.2Phosphoryl Group Transfers and ATP

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

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

27. Hydrolysis of ATP electrostatic repulsing • Hydrolysis, by causing charge separation (relieves electrostatic repulsing) • Pi is stabilized by formation of a resonance hybrid (same degree of double bound) • ADP2- immediately ionizes, releasing a proton into a medium of very low (H+). • Greater degree of solvation of the products Pi and ADP relative to ATP. solvation

28. Hydrolysis of ATP

30. Phosphorylation potential

31. Mg2+ and ATP • Forming of Mg2+ complexes partially shields the negative charges and influences the conformation of the phosphate groups in nucleotides such as ATP and ADP.

32. Hydrolysis of phosphoenolpyruvate (PEP) • Catalyzed by pyruvate kinase, this reaction is followed by spontaneous tautomerization of the product, pyruvate, tautomerization is not possible in PEP, and thus the products of hydrolysis are stabilized relative to reactants. Resonance stabilization of Pi also occurs.

33. Hydrolysis of 1, 3-bisphosphoglycerate • The direct product of hydrolysis is 3-phosphoglyceric acid, which has an un -dissociated carboxylic acid group, but dissociation occurs immediately. This ionization and the resonance structures, it makes possible stabilize the product relative to the reactants. • Resonance stabilization of Pi further contributes to the negative free-energy change.

34. Hydrolysis of phosphocreatine • Breakage of the P-N bond in phosphocreatine produces creatine, which is stabilized by formation of a resonance hybrid.

35. 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

36. Thioesters---Hydrolysis of acetyl-coenzyme A • Acetyl-CoA is a thioester with a large, negative, standard free energy of hydrolysis. Thioesters contain a sulfur atom in the position occupied by an oxygen atom in oxygen esters.

37. Free energy of hydrolysis for thioesters and oxygen esters • The products of both type of hydrolysis reaction have about the same free-energy content (G). Orbital overlap between the O and C atoms allows resonance stabilization in oxygen esters.

38. ATP provides energy by group transfers, Not by simple hydrolysis --- in two steps • A phosphoryl group is first transferred from ATP to glutamate • The phosphoryl group is displaced by NH3 and released as Pi • ATP can carry energy from high-energy phosphate compounds produced by catabolism to compounds such as glucose, converting them into more reactive species. • ATP thus serves as the universal energy currency in all living cells

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

40. High-energy compounds have a DG’o of hydrolysis more negative than -25 kJ/mol - 25 kJ/mol

41. Nucleophilic displacement reaction of ATP (SN2 nucleophilic displacements: PP67)– ATP donates 1).phosphoryl, 2).pyrophosphoryl, and 3).adenylyl groups

42. ATP as energy currency in many biochemistry reactions • Inorganic pyrophosphatase hydrolyed the PPi to two Pi, releasing 19 KJ/mol. • Activation of a fatty acid --- either for energy-yielding oxidation or for use in the synthesis of more complex lipids: is attached to the carrier coenzyme A. • Assembly of informational macromolecules – RNA . • ATP energizes active transport and muscle contraction. • Transphosphorylations between Nucleotides occur in all cell type (NTP—NDP: Nucleoside diphosphate kinase or Adenylate kinase or Creatine kinase, • Inorganic polyphosphate is a potential phosphoryl group donor (polyphosphate kinase —energy reservoir) Nucleoside diphosphate kinase Adenylate kinase Creatine kinase

43. Ping-Pong Mechanism of Nucleoside diphosphate The enzyme binds its first substrate (ATP), and a phosphoryl group is transferred to the side chain of a His residue. ADP departs, and another nucleoside diphosphate replace it, and this is converted to the corresponding triphosphate by transfer of the phosphoryl group from the phosphohistidine residue.

44. Fire flashes: glowing reports of ATP • From chemical energy into light energy. • An pyrophosphate cleavage of ATP to form luciferyl adenylate. In the presence of O2 and luciferase, the luciferin undergoes a multiple step oxidative decarboxylation to oxyluciferin and accompanied by remission of light.

45. 13.3Biological Oxidation- Reduction Reactions

46. Oxidation-reduction reactions Conjugated redox pair: Fe2+ (electron donor), and Fe3+ (electron acceptor)

47. Biological oxidations often involved dehydrogenation • The flow of electrons can do biological work. • O2 has a higher affinity of electrons—exergonic reaction— electromotive force (emf) provide energy to energy transducers (enzymes and other proteins) that do biological work. • When C share an electron pair with another atom. The sharing is unequal in favor of the more electronegative atom (H < C < S < N < O). • Electrons transfer in 4 ways: as e; as hydrogen atoms (H); as a hydride ion (H-); and combination with oxygen. • Reducing equivalent --- a single electron equivalent participating in an oxidation-reduction reaction--- biological oxidations as two reducing equivalents passing from substrate to oxygen.

48. 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 • Ared + Box Aox + Bred Reduced Coenzymes Conserve Energy from Biological Oxidations

49. 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 • Standard reduction potentialis for hydrogen: • Eo =H+ + e- ½ H2 Free-Energy Change Is Related to Reduction Potential

50. Measurement of the standard reduction potential (E’o) of a redox pair The ultimate reference half-cell is the hydrogen electrode, at pH 0. The electromotive force (emf) of this electrode is designated 0.00 V. At pH 7 in the test cell, E for the hydrogen electrode is 0.414 V. Electrons tend to flow through the external circuit from the half-cell of lower standard reduction potential to the half-cell of higher standard reduction potential. By convention, the half-cell with the stronger tendency to acquire electrons is assigned a positive value of E.