CHARACTERIZATION OF THE ZINC-BINDING SITE IN METHIONINE SYNTHASE ENZYMES OF ESCHERICHIA-COLI - THE ROLE OF ZINC IN THE METHYLATION OF HOMOCYSTEINE

X-ray absorption spectroscopy, using the analytical methodology descri bed in the previous paper, has been used to determine the ligation of the essential zinc ions in cobalamin-dependent (MetH) and cobalamin-in dependent (MetE) methionine synthases from Escherichia coil and to pro be directly the changes in zinc ligation that occur upon addition of t he thiol substrate, homocysteine, to each enzyme. Extended X-ray absor ption fine structure (EXAFS) spectra of native MetE and a truncated fr agment of MetH containing the substrate-binding sites are consistent w ith ZnS2(N/O)(2) and ZnS3(N/O) ligation, respectively. Previous mutage nesis studies of the homocysteine binding region of MetH had identifie d two putative thiolate zinc ligands, Cys310 and Cys311. Since the EXA FS spectra indicate that the zinc is coordinated to three sulfur ligan ds derived from the protein, a third conserved cysteine, Cys247, was m utated to alanine, resulting in a MetH fragment that binds only 0.09 e quiv of zinc per mol of protein and exhibits no methylcobalamin-homocy steine methyltransferase activity. Upon addition of L-homocysteine, th e X-ray absorption near edge structure changes for both enzymes, and t he EXAFS spectra show changes consistent with the coordination of a su lfur, giving a ZnS3(N/O) site for MetE and a ZnS4 site for MetH. Only the L-homocysteine enantiomer causes these effects; the addition of D- homocysteine to MetH(2-649) gives no detectable changes in the EXAFS o r the near edge regions. These results are consistent with a mechanism in which the homocysteine is ligated to zinc. Homocysteine is then ab le to initiate nucleophilic attack on the methyl group needed for meth ionine formation, with the methyl group bound either to methylcobalami n in MetH or to a polyglutamate derivative of methyltetrahydrofolate i n MetE.