Methylcobalamin as a substrate at a separate site on Escherichia coli B N5-methyltetrahydrofolate-homocysteine cobalamin methyltransferase

Abstract

Through the combined use of cellulose-polyacetate and disc-gel electrophoresis, a 200-fold purified preparation of Escherichia coli B 5-methyltetrahydrofolate-homocysteine methyltransferase was further fractionated into a single, protein-staining band. Associated with this band, as in the initial cobalamin (B 12) holoenzyme preparation, was a methyl-B 12-homocysteine methyltransferase activity. Several types of experiments support the view that 5-methyltetrahydrofolate-homocysteine transmethylation (Reaction 1) and methyl-B 12-homocysteine transmethylation (Reaction 2) occur at separate sites on the B 12-protein. Both sites are specific for a cobalt-methyl group, however. An inhibited (Reaction 1) propyl-B 12 enzyme catalyzed Reaction 2 with no exchange or decomposition of the propyl-B 12 group. Reconstituted holoenzyme containing a tritiated B 12 group catalyzed Reaction 2 with little loss of its tightly bound chromophore. Aquo-B 12, but not propyl-B 12, markedly inhibited Reaction 2. Distinctly different pH-activity curves were observed for the two activities. The apparent K m of urea-resolved apoenzyme for methyl-B 12 as a prosthetic group (Reaction 1) was 2.0 μ m; whereas, the K m of holoenzyme for methyl-B 12 as a substrate (Reaction 2) was >2.5 m m. At a methyl-B 12 concentration of 2.5 m m Reaction 2 was catalyzed at approximately the rate of Reaction 1.