Equilibrium Constants of the Malate Dehydrogenase, Citrate Synthase, Citrate Lyase, and Acetyl Coenzyme A Hydrolysis Reactions under Physiological Conditions

Abstract The observed equilibrium constants (Kobs) of the malate dehydrogenase (EC 1.1.1.37), citrate synthase (EC 4.1.3.7) and citrate lyase (EC 4.1.3.6) reactions have been determined under near physiological conditions (38°, pH 7.0, I = 0.25, free [Mg2+] = 10-3 m). From these values the observed standard free energy change (ΔG0obs) for the hydrolysis of acetyl-CoA has been determined. Under the above conditions, and taking the standard state of liquid water to have activity = unity ([H2O] = 1), the equilibrium constants of the three reactions at pH 7.0 may be written: Malate dehydrogenase Kobs = [Oxaloacetate][NADH]/[Malate][NAD+] = 2.86 ± 0.12 x 10-5 Citrate synthase Kobs = [Citrate][CoA]/[Oxaloacetate][Acetyl-CoA][H2O] = 2.24 ± 0.11 x 106 Citrate lyase Kobs = [Citrate]/[Oxaloacetate][Acetate] = 2.22 ± 0.16 m-1 where the concentration of each substrate represents the total of all the ionic species present, both acid forms and magnesium complexes. The values obtained for Kobs of the citrate synthase and citrate lyase reactions vary to the same extent with the changes in magnesium concentration. At free [Mg2+] = 0, Kobs for the citrate synthase reaction is 1.01 ± 0.05 x 106 and the Kobs for the citrate lyase reaction is 1.00 ± 0.07 m-1. In contrast, malate dehydrogenase is unaffected by the concentration of free Mg2+ up to 4 mm. From the constants of the citrate synthase and citrate lyase reactions, the Kobs for the hydrolysis of acetyl-CoA under near physiological conditions is calculated to be 1.01 x 106 m, corresponding to a free energy change of -8.54 Cal per mole (-35.75 kJ per mole) independent of the free magnesium concentration.