Escherichia coli B N5-methyltetrahydrofolate-homocysteine cobalamin methyltransferase: Activation with S-adenosyl-l-methionine and the mechanism for methyl group transfer

Abstract E. coli B cobalamin methyltransferase was activated upon treatment with S-adenosyl- l -methionine (AMe). Activation was attributed for several reasons to the conversion of the original cobalamin enzyme to a methylcobalamin enzyme. It resulted only when the cobalamin enzyme was incubated with AMe under the exact same reducing conditions which yield a methyl-14C-cobalamin enzyme with methyl-14C-AMe and it was prevented by propylation of the cobalamin prosthetic group. Activation was also achieved with methyl iodide. Furthermore, the ability of an activated cobalamin enzyme to synthesize methionine aerobically was lost if it was exposed to either tetrahydrofolate or homocysteine at 37 ° prior to an incubation with N5-methyl-14C-tetrahydrofolate (N5-methyl-14C-H4-folate) plus homocysteine. This inactivation was correlated with the demethylation of a methyl-14C-cobalamin enzyme which had been prepared with methyl-14C-AMe. A methylcobalamin enzyme, freed of unbound AMe, catalyzed limited N5-methyl-H4-folate-homocysteine transmethylation in the absence of reduced flavin. Based on the total amount of bound cobalamin (ca. 0.4 mμmole) a minimum turnover of 8–10 fold was observed aerobically. Methionine synthesis was limited under aerobic conditions by the rapid inactivation of the cobalamin enzyme during catalysis. The total turnover by 0.05–0.1 mμmole of methylcobalamin enzyme was increased to about 100-fold when the reaction mixtures were incubated either in a vacuum or else under nitrogen gas. Exogenous AMe did not improve the amount of catalysis by a methylcobalamin enzyme when reduced flavin was absent. The original, unmethylated cobalamin enzyme did not display the limited catalytic activity which was observed with a methylcobalamin enzyme. In a flavin-reducing system AMe was essential for 1.0 μμmole of both the methyl cobalamin enzyme and the original cobalamin enzyme to maintain a constant rate of N5-methyl-H4-folate-homocysteine transmethylation. When 1.8 mμmoles of unlabeled methylcobalamin enzyme were incubated with an excess of N5-methyl-14C-H4-folate and 0.1–0.4 mμmole of homocysteine (added simultaneously) the resulting methyl-14C-methionine had a much lower specific activity than the substrate methyl-14C-donor. The synthesized methyl-14C-methionine had the same specific activity, however, if the unlabeled methylcobalamin enzyme was incubated first with N5-methyl-14C-H4-folate and then with 0.1–0.4 mμmole of homocysteine. By this prior treatment with N5-methyl-14C-H4-folate, methyl-14C groups were exchanged onto the cobalt atom without appreciably inactivating the enzyme for subsequent aerobic methyl-14C-methionine synthesis. Additional catalytic properties of the cobalamin methyltransferase were studied. The enzyme was observed to catalyze a methyl group exchange reaction between pteridine ring labeled N5-methyl-H4-folate and unlabeled H4-folate. Exchange trans-methylation required AMe as a cofactor but was not dependent upon reduced flavin because the high concentration of H4-folate which was employed as a substrate also apparently functioned as a reducing agent for the cobalamin enzyme. This conclusion was based upon the finding that H4-folate can be substituted for reduced flavin and permit methyl-14C-methionine synthesis from N5-methyl-14C-H4-folate plus homocysteine at 1 3 the rate attainable in the presence of reduced flavin. Catalysis of methyl group transfer from AMe to H4-folate was demonstrated and in this reaction too, reduced flavin was not essential. H4-Folate (1.5 m m ) inhibited by 2.4-fold the rate of N5-methyl-H4-folate-homocysteine transmethylation in the presence of reduced flavin; however, it increased by 2.4–3.0-fold the rate of AMe-homocysteine transmethylation in the presence of reduced flavin. Chemical propylation of the cobalamin prosthetic group inhibited equally and in a light reversible manner methyl group transfer from AMe to H4-folate; from N5-methyl-H4-folate to homocysteine; and from AMe to homocysteine (± H4-folate). Additional data were also collected on the formation and breakdown of a methyl-14C-eobalamin enzyme. Within the first minute of incubation at 37 ° the rate of AMe-dependent methyl-14C-cobalamin enzyme formation from N5-methyl-14C-H4-folate was increased by 4–5-fold when limiting amounts of either homocysteine or H4-folate were added to the system. Contrary to what was previously observed with a 1:1 ratio of active N5-methyl-14C-H4-folate to cobalamin enzyme, dithiothreitol was not required in order to obtain maximal amounts of methyl-14C-cobalamin enzyme when a 10:1 ratio was used. Nevertheless, the rate of methyl-14C-cobalamin enzyme formation was slower in the absence of dithiothreitol, unless limiting quantities of homocysteine were present. Demethylation of a methyl-14C-cobalamin enzyme with homocysteine was biphasic. It consisted of an initial rapid reaction which was complete in less than 5 sec at 37 ° and a very slow reaction which spanned the next 15 min at 37 °. About 70% of the methyl-14C-groups were transferred to homocysteine within the first 5 sec at a rate which was too rapid to measure. On the basis of the foregoing results and other recent work, a reaction mechanism for N5-methyl-H4-folate-homocysteine transmethylation is suggested and discussed in detail. In the reaction scheme which is proposed methyl groups from N5-methyl-H4-folate are transferred to homocysteine via a methylcobalamin enzyme intermediate. AMe is assigned the possible role of an “activator” which methylates only inactive forms of the cobalamin protein, as, for example, the cobalamin enzyme species which one isolates from cell-free extracts.