Cobalamin-independent methionine synthase from Escherichia coli: a zinc metalloenzyme.

Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine. Previous work had shown the existence of a reactive thiol group, cysteine 726, whose alkylation led to loss of all detectable enzymatic activity [González, J.C., et al. (1992) Biochemistry 31, 6045-6056]. A site-directed mutation of MetE, Cys726Ser, was constructed to investigate the possible role of this cysteine. The Cys726Ser protein was purified to homogeneity, affording a protein with no detectable activity. To assess the possibility that cysteine726 functions as a metal ligand, inductively coupled plasma-atomic emission spectrometry was performed. The wild-type enzyme contains 1.02 equiv of zinc per subunit; the Cys726Ser mutant does not contain zinc, supporting the view that cysteine726 is required for metal binding. A loss of enzymatic activity is observed upon removal of zinc from the wild-type MetE by incubation in urea and EDTA; activity can subsequently be restored by zinc reconstitution, suggesting that zinc is required for catalysis. Circular dichroism measurements further suggest that there are no major differences in the secondary structures of the wild-type and the Cys726Ser mutant enzymes. Extended X-ray absorption fine structure analysis has established that the average zinc environment is different in the presence of homocysteine than in its absence and is consistent with the changes expected for displacement of an oxygen or nitrogen ligand by the sulfur of homocysteine. A possible model for zinc-dependent activation of homocysteine by MetE is presented.