The amino acid specificity of Bacillus stearothermophilus tyrosyl-tRNA synthetase was studied by site-directed mutagenesis of residues close to the active site. X-ray crystallographic studies of the enzyme have suggested that Asp-176 is a major determinant of amino acid specificity, as its carboxylate is observed to make a hydrogen bond with the hydroxyl group of the substrate tyrosine. Previous efforts to test the importance of Asp-176 by site-directed mutagenesis led to inactive enzymes. We have now investigated the catalytic properties of enzymes altered, not at Asp-176 itself, but instead at two amino acids, Asn-123 and Trp-126, that appear in the crystallographic structure to form hydrogen bonds with Asp-176. Mutation of Trp-126 does not affect the kinetics of activation with respect to ATP but leads to modest increases in the Km for tyrosine. Conversely, position Asn-123 mutants are strongly affected: 160-fold lower kcat and 5-fold higher Km for the Ala-123; and 17-fold decrease and 270-fold increase, respectively, of the same parameters for the Asp-123 mutation. The specificity against phenylalanine was determined from the ratios of kcat/Km for the amino acids in the pyrophosphate exchange reaction. The ratio of 1.2 x 10(5) for the wild-type enzyme decreases 4-fold on mutation of Asn-123 but increases 7-fold on the mutation of Trp-126-->Phe and 2-fold on Trp-126-->Leu. The wild-type enzyme has not reached the maximum limit of discrimination between tyrosine and phenylalanine.