Equilibrium constants under physiological conditions for the reactions of d-3-phosphoglycerate dehydrogenase and l-phosphoserine aminotransferase

The observed equilibrium constants ( K obs) for the reactions of d-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) and l-phosphoserine aminotransferase (EC 2.6.1.52) (the first two steps of l-serine biosynthesis) have been determined at 38 °C, under conditions of physiological ionic strength (0.25 m) and physiological ranges of pH and magnesium concentrations. From these observed constants and the acid dissociation and metal binding constants of the substrates, an ionic equilibrium constant ( K) has been calculated for each reaction. Using Σ and square brackets to indicate total concentrations, for the reaction of d-3-phosphoglycerate dehydrogenase, K obs = [Σ phosphohydroxypyruvate][Σ NADH] [Σ D-3-phosphoglycerate][Σ NAD + ] , K = [ phosphohydroxypyruvate 3− ][ NADH]( H + ) [ D-3-phosphoglycerate 3− ][ NAD + ] ; and for the reaction of l-phosphoserine aminotransferase, K obs = [Σ α- ketoglutarate][Σ L-phosphoserine] [Σ phosphohydroxypyruvate][Σ L-glutamate] , K = [α- ketoglutarate 2− ][ L-H · phosphoserine 2− ] [ phosphohydroxypyruvate 3− ][ L-H · glutamate − ] The values of K for the reaction of d-3-phosphoglycerate dehydrogenase are 3.45 ± 0.04 × 10 −13 m (38 °C) [ΔG ° = +74.2 kJ/mol (+17.7 kcal/mol)] and 1.30 ± 0.01 × 10 −13 m (25 °C) [ΔG ° = +73.5 kJ/mol (+17.6 kcal/mol)]. K obs at 38 °C, pH 7.0, and [K +] = 0.2 m is 2.92 × 10 −6 ([Mg 2+] = 0) and 2.89 × 10 −6 ([Mg 2+] = 1 m m). The value of K for the reaction of l-phosphoserine aminotransferase is 133 ± 1 [ΔG 0 = −12.5 kJ/mol (−3.0 kcal/mol)] a value insignificantly affected by temperature (4–43 °C). K obs at pH 7.0 (38 °C, [K +] = 0.2 m) is 85.9 ([Mg 2+] = 0), 84.6 ([Mg 2+] = 1 m m) and 80.7 ([Mg 2+] = 10 m m). For these calculations the acid dissociation and potassium and magnesium binding constants of d-3-phosphoglycerate, phosphohydroxypyruvate, and l-phosphoserine were determined under physiological conditions by titration. The values of the equilibrium constants reported in this work are highly significantly different from direct determinations reported by others. The implications of the results to the understanding of the biosynthesis of l-serine are discussed.