Studies on cobalamin-independent methionine synthase from Escherichia coli: A zinc metalloenzyme.

Abstract

Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine. Two families of alkylating agents were found to inhibit MetE. Treatment of MetE with these agents results in a time- and concentration-dependent loss of enzymatic activity. Iodoacetamide results in a faster and more complete inactivation than iodoacetic acid, and 1-tosylamido- scL-phenylalanyl chloromethylketone (TPCK) acts at a faster rate than 1-tosylamido- scL-lysyl chloromethylketone (TLCK). Using $\sp{14}$C-iodoacetamide, it was determined that inactivation results from covalent modification of the enzyme and that alkylation takes place almost exclusively on cysteine726. Pre-treatment of MetE with TPCK prevents protein alkylation by iodoacetamide, suggesting that TPCK also reacts with cysteine726. Using the method of site-directed mutagenesis by overlap-extension PCR, a site-directed mutant of MetE, Cys726Ser, was constructed to investigate the possible role of this cysteine. The Cys726Ser protein can be purified to homogeneity, affording a protein with no detectable activity. Inductively coupled plasma-atomic emission spectrometry was performed on samples of wild-type and Cys726Ser mutant enzyme. These studies demonstrated that the wild-type enzyme contains 1.02 equivalents of zinc per subunit; the Cys726Ser mutant, however, does not contain zinc, suggesting 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 suggest that there are no significant differences in the secondary structures of the wild-type and the Cys726Ser mutant enzymes that would account for the differences in enzymatic activity, suggesting a non-structural role for zinc. Extended X-ray absorption fine structure (EXAFS) data suggest binding of homocysteine to the zinc center. These results are consistent with a model in which zinc activates the homocysteine substrate for nucleophilic attack on methyltetrahydrofolate in cobalamin-independent methionine synthase.