Figure 2.

Effect of inorganic dianions and Gua⁺ on R269A mutant hlGPDH-catalyzed reduction of GA by NADH, determined for reactions at pH 7.5, 25 °C, saturating [NADH] = 0.2 mM and I = 0.12 (NaCl). (A) The increase in v/[E][GA] (M^–1 s^–1), with increasing [HPO₃^2–], for reactions in the presence of 30 mM (open symbols) or 60 mM (closed symbols) total GA [carbonyl + hydrate] and at different fixed concentrations of Gua⁺. The equilibrium constant for hydration of GA is K_eq = [carbonyl]/[hydrate] = (6/94).⁸ Key: ([GA]_carbonyl = 3.6 mM) (▼) 30 mM Gua⁺; (⧫), 25 mM Gua⁺; (■), 20 mM Gua⁺; (▲), 15 mM Gua⁺; (●), 10 mM Gua⁺; ([GA]_carbonyl = 1.8 mM) (□), 30 mM Gua⁺; (◊), 15 mM Gua⁺. (B) The increase in v/[E][GA] (M^–1 s^–1), with increasing [FPO₃^2–], for reactions at [GA]_carbonyl = 3.6 mM and at different fixed concentrations of Gua⁺. Key: (▼) 30 mM Gua⁺; (⧫), 25 mM Gua⁺; (■), 20 mM Gua⁺; (▲), 15 mM Gua⁺; (●), 10 mM Gua⁺. (C) The increase in v/[E][GA] (M^–1 s^–1), with increasing [SO₄^2–], for reactions at 3.6 mM GA and at different fixed concentrations of Gua⁺. Key: (▼) 30 mM Gua⁺; (⧫), 25 mM Gua⁺; (■), 20 mM Gua⁺; (▲), 15 mM Gua⁺; (●), 10 mM Gua⁺. Figure 3 shows the linear plots of the slopes of these linear correlations, (k_cat/K_GAK_X)_obs, against [Gua⁺].