Escherichia coli B N5-methyltetrahydrofolate-homocysteine cobalamin methyltransferase: gel-filtration behavior of apoenzyme and holoenzymes

Abstract

1. 1. Sephadex G-200 chromatography revealed a significant difference in the gel-filtration behavior of urea-resolved apomethyltransferase compared to three forms of the enzyme containing bound cobalamin (B 12). The initial (unalkylated) B 12 holoenzyme, a cobalt-propyl-B 12 enzyme, and reconstituted methyl-B 12 holoenzyme each eluted as if their molecular weights were 150 000; whereas, urea-resolved apoenzyme appeared to have a molecular weight of 205 000. In addition, the following average Stokes radii were obtained: initial B 12 holoenzyme, 54.2 Å; urea-resolved apoenzyme, 62.9 Å; reconstituted methyl-B 12 holoenzyme, 53.6 Å; and propyl-B 12 enzyme, 55.1 Å. Sephadex G-200 chromatography of apoenzyme in the presence of 6.0 M urea + 10 mM 1,4-dithiothreitol indicated that dissociation into subunits is not essential in order to remove the B 12 from holoenzyme with urea.