Purification and Characterization of Nicotinamide Adenine Dinucleotide-dependent Methylenetetrahydrofolate Dehydrogenase from Clostridium formicoaceticum

Methylenetetrahydrofolate dehydrogenase from the obligate anaerobic mesophile Clostridium formicoaceticum has been purified to homogeneity. It has a molecular weight of 60,000 ± 3,000 and consists of two subunits of equal molecular weight. The sedimentation constant s020, w is 3.8 S, the partial specific volume is 0.74 ml per g, and the Stokes radius is 33.2 A. The enzyme is specific for NAD, and with NADP there is no activity. NADP is only slightly inhibitory at concentrations above 10-2 m. The pure enzyme has an activity of 1,400 µmoles min-1 mg-1 at 37°, pH 7. It is active only with l, l-5,10-methylenetetrahydrofolate, and the d, l-isomer does not affect the reaction. The apparent Km for NAD is 7.9 x 10-4 m and for l, l-5,10-methylenetetrahydrofolate 6.6 x 10-5 m at 37° in 0.2 m potassium maleate, pH 7. The temperature optimum is about 45°. Below this temperature the enzyme exhibits a linear relationship in an Arrhenius plot, and the apparent activation energy is 8,500 cal mole-1.