N5-methyltetrahydrofolate-homocysteine transmethylase. Propylation characteristics with the use of a chemical reducing system and purified enzyme.

A simple method is described whereby millimicromole amounts of extensively purified N5-methyltetrahydrofolatehomocysteine transmethylase (vitamin Blz transmethylase) can be chemically propylated to yield an inhibited vitamin Blz 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 prop~l-l-*~C 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 propyfation on four separate transmethylation reactions catalyzed by the vitamin Blz 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-Bj2 ($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 iV6-methyltetrahydrofolate a noninhibitory amount of AMe was essential.