Escherichia coli B 5-methyltetrahydrofolate-homocysteine cobalamin methyltransferase: Resolution and reconstitution of holoenzyme

Abstract

Extensively purified E. coli B cobalamin methyltransferase was resolved into an apoenzyme plus an unbound, 1-electron-reduced, cobalamin (B 12r) upon incubation for 15 min at 37 ° with 6.0 m urea containing dithiothreitol (DTT). Apoenzyme which had been prepared by urea resolution was reconstituted 75–80% into a radioactive holoenzyme complex upon a 5-min incubation at 37 ° with 10 nmoles of either methyl- 14CB 12 or propyl- 14CB 12 per mg of protein. Reconstituted methyl- 14CB 12 enzyme was light-stable (100 W tungsten lamp, 0 °) unless acidified and was almost completely demethylated by homocysteine. Incubation of resolved apoenzyme with deoxyadenosyl-B 12, hydroxy-B 12, cyano-B 12, B 12r, diaquocobinamide, and methylcobinamide resulted in negligible holoenzyme formation. However, under the same conditions sulfito-B 12 yielded 48–52% holoenzyme. Urea-prepared apoenzyme differed from the purified B 12 holoenzyme with respect to its sedimentation coefficient. Based on a sedimentation coefficient of 7.0 S for the purified B 12 holoenzyme the apoenzyme sedimented at a slightly slower rate with a relative coefficient equal to 6.2 S. Binding methyl-B 12( 3H) to the apoenzyme yielded a reconstituted methyl-B 12( 3H) holoenzyme with a sedimentation coefficient of 7.0 S. Unpurified holoenzyme and unpurified apoenzyme in cell-free, sonic extracts also centrifuged differently, like purified holoenzyme and urea-resolved apoenzyme, respectively.