The reaction parameters for methyl-14C-cobalamin enzyme formation with methyl-14C-S-adenosyl-l-methionine (methyl-14C-AMe) were determined and the relationship of this methylation reaction to the AMe-dependent formation of a methyl-14C-cobalamin enzyme with N5-methyl-14C-tetrahydrofolate (N5-methyl-14C-H4-folate) was studied. Incubation of cobalamin methyltransferase either at 37 ° with methyl-14C-AMe alone or at 0 ° with methyl-14C-AMe plus unlabeled N5-methyl-H4-folate yielded about 1 equivalent of bound methyl-14C-cobalamin per equivalent of enzyme-bound cobalamin. A flavin reducing system was essential for methylation at both temperatures. The 37 ° methylation by methyl-14C-AMe was essentially complete after 1 min; whereas, the 0 ° methylation required 15-20 min and was negligible during the first 3 min. As expected, the yield of methyl-14C-cobalamin enzyme decreased markedly when the 0 ° methylation mixture was incubated at 37 ° because at the higher temperature the 0 ° system was converted to the corresponding system which has been employed routinely for the AMe-dependent methylation by N5-methyl-14C-H4-folate. Time studies at 37 ° throughout 6 min of incubation revealed that the cobalamin enzyme was first methylated by AMe within 60 sec and then methylated by N5-methyl-H4-folate over the next 6 min. A methyl-14C-cobalamin enzyme which had been prepared initially in a flavin reducing system was found to exchange its methyl-14C group with the methyl group of unlabeled N5-methyl-H4-folate. This exchange occurred aerobically and yielded an unlabeled methylcobalamin enzyme plus enzymatically active N5-methyl-14C-H4-folate. Alternatively, a methyl-14C enzyme could transfer its methyl group to H4-folate yielding the active isomer of N5-methyl-14C-H4-folate. With the use of three differently labeled 14carbon AMe's, several equivalents of this cofactor per equivalent of bound cobalamin were observed to bind tightly and non-covalently to protein in the enzyme preparations. A flavin reducing system was not required for the binding and this noncovalently bound AMe was not essential for methylation with N5-methyl-14C-H4-folate. Methyl iodide could be substituted for AMe and permitted methylation of the cobalamin enzyme with N5-methyl-14C-H4-folate. Formation of a methyl-14C-cobalamin enzyme with N5-methyl-14C-H4-folate (triglutamate) also required AMe and reduced flavin. Cobalamin enzyme preparations which were only partially dependent on AMe for methylation by N5-methyl-14C-H4-folate became completely dependent on AMe if they were first incubated with homocysteine in the presence of reduced flavin and dithiothreitol and then reisolated. Quantitatively, the amount of H4-folate which was formed upon methylation of the cobalamin enzyme with N5-methyl-14C-H4-folate was much less than the amount of methyl-14C-cobalamin enzyme which accumulated in the same incubation mixtures. Collectively, the results indicate that in our reaction system methylation of the bound cobalamin by AMe is a prerequisite to its methylation by N5-methyl-14C-H4-folate. Methylation by the latter is mediated by a methyl group exchange in which traces of H4-folate could function catalytically. © 1969.
