N5-Methyltetrahydrofolate-Homocysteine Transmethylase

A simple method is described whereby millimicromole amounts of extensively purified N5-methyltetrahydrofolate-homocysteine transmethylase (vitamin B12 transmethylase) can be chemically propylated to yield an inhibited vitamin B12 enzyme which is reactivated with light. The procedure consists of a short incubation of the enzyme with propyl iodide in the presence of reduced flavin mononucleotide and dithiothreitol under hydrogen gas. Formation of a propylated (inhibited) enzyme was correlated with alterations in the visible absorption spectrum of the enzyme. When propyl-1-14C bromide was the alkylating agent a radioactive enzyme was formed which lost radioactivity upon exposure to light. The effects of several reaction parameters on the extent of propylation were examined as was the effect of propylation on four separate transmethylation reactions catalyzed by the vitamin B12 enzyme preparations. Transmethylation reactions which require catalytic amounts of S-adenosylmethionine (AMe) as a cofactor or in which AMe itself is the substrate methyl donor were inhibited by propylation; however, methyl group transfer from methyl-B12 (5,6-dimethylbenzimidazolylcobamide methyl) to homocysteine was not affected by propylation. Micromolar concentrations of both AMe and N5-methyltetrahydrofolate markedly inhibited propylation as did higher levels of methyl iodide. For the inhibition of propylation by low concentrations of N5-methyltetrahydrofolate a noninhibitory amount of AMe was essential.