K. Peariso, C. Goulding, Shan Huang
Aug 11, 1998
Citations
3
Influential Citations
90
Citations
Quality indicators
Journal
Journal of the American Chemical Society
Abstract
X-ray absorption spectroscopy, using the analytical methodology described in the previous paper, has been used to determine the ligation of the essential zinc ions in cobalamin-dependent (MetH) and cobalamin-independent (MetE) methionine synthases from Escherichia coli and to probe directly the changes in zinc ligation that occur upon addition of the thiol substrate, homocysteine, to each enzyme. Extended X-ray absorption fine structure (EXAFS) spectra of native MetE and a truncated fragment of MetH containing the substrate-binding sites are consistent with ZnS2(N/O)2 and ZnS3(N/O) ligation, respectively. Previous mutagenesis studies of the homocysteine binding region of MetH had identified two putative thiolate zinc ligands, Cys310 and Cys311. Since the EXAFS spectra indicate that the zinc is coordinated to three sulfur ligands derived from the protein, a third conserved cysteine, Cys247, was mutated to alanine, resulting in a MetH fragment that binds only 0.09 equiv of zinc per mol of protein and exhibi...