The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc. This metal ion is strongly bound, as it is not removed by 8 M urea. Slow removal of the zinc at 4 degrees C in the presence of the specific chelating agent, 1,10-phenanthroline, is proportional to the loss of aminoacylation activity and to the presence of a more open conformer of the enzyme. This conformer migrates more slowly than the native enzyme during gel electrophoresis under nondenaturing conditions and binds tRNA(Glu). Infrared spectroscopy measurements show that it differs from the native enzyme by a lower alpha-helix content and a higher proportion of beta-sheet and unordered structures. ATP protects the enzyme against 1,10-phenanthroline-mediated zinc removal, suggesting that the zinc-binding region is closely associated with the catalytic site. Additional support for this conclusion comes from the presence of zinc in the 27-kDa N-terminal half of the enzyme and in a 10-kDa fragment. The latter is homologous to the tRNA acceptor helix binding domain of E. coli glutaminyl-tRNA synthetase. The presence of the conserved CYC motif in this domain of the zinc-containing glutamyl-tRNA synthetases of E. coli and Bacillus subtilis, and its absence in that of Thermus thermophilus and the E. coli glutaminyl-tRNA synthetase which do not contain zinc, suggest that the cysteines of this motif and the C- and H-rich 125CRHSHEHHX5C138 segment present in the 10-kDa zinc-binding fragment are involved in zinc binding by the glutamyl-tRNA synthetase of E. coli.