Approaches to Metabolism

5. Approaches to Metabolism • PATHWAYS • ENERGETICS (THERMODYNAMICS) • REGULATION • CELLULAR FUNCTION / LOCALIZATION • ENZYME MECHANISM • MECHANISM

6. Introduction to Metabolism • Understanding metabolic pathways • for each step: • reaction mechanism including enzyme participation • thermodynamics --- “will it go”? • contribution to whole pathway

7. THERMODYNAMICS IN BIOCHEMISTRY • SYSTEM: defined region •  SURROUNDINGS: the rest of the universe • FIRST LAW OF THERMODYNAMICS The total energy of a system and its surroundings is a constant

8. THERMODYNAMICS EA energy in a system at the start of a process EB energy in a system at the end of a process Q Heat absorbed by the system from its surroundings W Work done by the system Note: path-independent

9. Second Law of Thermodynamics Entropy (S): the degree of randomness or disorder in a system A process can occur spontaneously only if the sum of the entropies of the system and its surroundings increases

11. Entropy • Does it predict spontaneous reactions? _________________ •  Problems • difficult to measure • must determine Ssystem and Ssurroundings

12. Gibbs Free Energy • G Gibbs Free Energy • H Change in enthalpy (heat content) • S Change in entropy • T Temperature

14. Gibbs Free Energy • G Change in (Gibbs) free energy is the indicator of the reaction; (G) is heat change (H ) modified by T and change in freedom (entropy) S; G is the useful (non- heat) energy generated & absorbed in biological reactions • H Change in enthalpy (heat content); measured in biological reactions as heat generated (exothermic - H) or heat absorbed (endothermic + H) • S Change in entropy • T Temperature

15. Gibbs Free Energy G < 0 A reaction can occur spontaneously G = 0 A system is at equilibrium: no net change occurs G > 0 A reaction cannot occur spontaneously. An input of free energy is required to drive the reaction.

17. Gibbs Free Energy • G is path-independent • G provides no information about rates of (enzyme-assisted) reactions

18. DG and Equilibrium For the reaction A + BC + D

19. DG and Equilibrium Go standard free energy change at pH 0 (a H+ conc. of 1.0 M) T = 2980 K is equal to 250C   concentration of all reactants is 1.0 M R = 1.98 kcal mol-1 deg-1

20. DG and Equilibrium • This equation relates the nature of the reactants and their concentrations. • Gº' is the standard free energy change for biochemical reactions • pH = 7 (H+ = 10-7 M) and activity of water (55.6 M) • Concentration of all reactants = 1.0 M ’

21. Equilibrium Constants  Keq equilibrium constant G = Go’ + R T lnKeq At equilibrium G = 0, then Go’ = -R T lnKeq

22. Equilibrium Constants  Keq equilibrium constant G = Go’ + R T lnKeq Keq Go’ (kJ/mol) 99/1 -10 1/99 +10 105/1 -30 1/105 +30

23. Equilibrium Constants = - 2.303RT log10 Keq Keq = 10- DG°’/(2.303 RT) Substitute R = 1.987 x 10-3 kcal mol-1 deg-1 T = 2980 K (corresponding to 250C) Keq = 10- DG°’/1.36

24. Determination of DG°’ DHAP  Glyceraldehyde-3-phosphate • At equilibrium, 298K, pH 7, the ratio of [G-3-P]/[DHAP] = 0.0475. •  At equilibrium, by definition G = 0

25. Determination of DG°’ DHAP  Glyceraldehyde-3-phosphate (GAP) Gº' = - RT ln(0.0475) Gº' = - 2.303RT log10 Keq = -2.303 x 1.98 x 10-3 kcal mol-1 deg-1 x 298 x log10(0.0475) = + 1.8 kcal/mol

26. Determination of DG DG, i.e. not at equilibrium: DHAP (2 x 10-4 M);GAP (3 x 10-6 M) G = Go’ + R T lnKeq G = Gº' + 2.303RT log10 Keq = 1.8 kcal mol-1 + 2.303 RT log10 3 x 10-6 2 x 10-4 = 1.8 kcal mol-1 - 2.49 kcal mol-1 = -0.69 kcal mol-1 (-2.89 kJ mol-1); 1 kcal = 4.184 kJ

27. USING Gº' TO DETERMINE [REACTANTS] What are the equilibrium concentrations of fructose-1,6-bisphosphate, dihydroxyacetone phosphate, and glyceraldehyde 3-phosphate when 1 mM fructose 1,6-bisphosphate is incubated with aldolase under standard conditions? See Stryer 5e, chapter 16!