FIGURE 4.

Redox cofactor specificity of MTHFD2L at physiological concentrations of P_i, Mg²⁺, and 5,10-CH₂-THF. The ratio of NAD⁺- to NADP⁺-dependent 5,10-methylene-THF activity was plotted as a function of increasing concentrations of inorganic phosphate (A), Mg²⁺ (B), or 5,10-CH₂-THF (C). Reaction buffer (described in the legend for Fig. 3) contained either 1 mm NAD⁺ or 6 mm NADP⁺. 5,10-CH₂-THF was included at 0.4 mm (A and B) or varied (C). For dependence on P_i concentration (A), 5 mm MgCl₂ was included for both NAD⁺- and NADP⁺-dependent reactions. For dependence on Mg²⁺ concentration (B), 25 mm potassium phosphate was included in NAD⁺-dependent but not NADP⁺-dependent reactions. For dependence on 5,10-CH₂-THF concentration, 5 mm MgCl₂ and 25 mm P_i were included in NAD⁺-dependent reactions. For the NADP⁺-dependent reactions, only 5 mm MgCl₂ was included. Reported mitochondrial matrix substrate concentration ranges (indicated by shaded areas) are 5–15 mm for P_i (47–49), 0.3–0.4 mm for Mg²⁺ (48, 49), and 2.5–25 μm for 5,10-CH₂THF (37–40), respectively. The best estimates found for in vivo mitochondrial NADP⁺ and NAD⁺ concentrations are NADP⁺ = 80 μm and NAD⁺ = 240 μm (42, 43). The data in C were fit to the Michaelis-Menten equation. The 5,10-CH₂-THF concentration that gave the half-maximal ratio of NAD⁺- to NADP⁺-dependent 5,10-methylene-THF activity was 12.5 μm.