Eschericha coli B N5-methyltetrahydrofolate-homocysteine methyl-transferase: Sequential formation of bound methylcobalamin with S-adenosyl-l-methionine and N5-methyltetrahydrofolate

Abstract The reaction parameters for methyl- 14 C-cobalamin enzyme formation with methyl- 14 C-S-adenosyl- l -methionine (methyl- 14 C-AMe) were determined and the relationship of this methylation reaction to the AMe-dependent formation of a methyl- 14 C-cobalamin enzyme with N 5 -methyl- 14 C-tetrahydrofolate ( N 5 -methyl- 14 C-H 4 -folate) was studied. Incubation of cobalamin methyltransferase either at 37 ° with methyl- 14 C-AMe alone or at 0 ° with methyl- 14 C-AMe plus unlabeled N 5 -methyl-H 4 -folate yielded about 1 equivalent of bound methyl- 14 C-cobalamin per equivalent of enzyme-bound cobalamin. A flavin reducing system was essential for methylation at both temperatures. The 37 ° methylation by methyl- 14 C-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- 14 C-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 N 5 -methyl- 14 C-H 4 -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 N 5 -methyl-H 4 -folate over the next 6 min. A methyl- 14 C-cobalamin enzyme which had been prepared initially in a flavin reducing system was found to exchange its methyl- 14 C group with the methyl group of unlabeled N 5 -methyl-H 4 -folate. This exchange occurred aerobically and yielded an unlabeled methylcobalamin enzyme plus enzymatically active N 5 -methyl- 14 C-H 4 -folate. Alternatively, a methyl- 14 C enzyme could transfer its methyl group to H 4 -folate yielding the active isomer of N 5 -methyl- 14 C-H 4 -folate. With the use of three differently labeled 14 carbon 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 N 5 -methyl- 14 C-H 4 -folate. Methyl iodide could be substituted for AMe and permitted methylation of the cobalamin enzyme with N 5 -methyl- 14 C-H 4 -folate. Formation of a methyl- 14 C-cobalamin enzyme with N 5 -methyl- 14 C-H 4 -folate (triglutamate) also required AMe and reduced flavin. Cobalamin enzyme preparations which were only partially dependent on AMe for methylation by N 5 -methyl- 14 C-H 4 -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 H 4 -folate which was formed upon methylation of the cobalamin enzyme with N 5 -methyl- 14 C-H 4 -folate was much less than the amount of methyl- 14 C-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 N 5 -methyl- 14 C-H 4 -folate. Methylation by the latter is mediated by a methyl group exchange in which traces of H 4 -folate could function catalytically.