MODIFICATION OF THE AMINO-ACID SPECIFICITY OF TYROSYL-TRANSFER-RNA SYNTHETASE BY PROTEIN ENGINEERING

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-1 76 itself, but instead at two amino acids, Asn- 1 23 and Trp- 126, that appear in the crystallographic structure to form hydrogen bonds with Asp- 1 76. Mutation of Trp-126 does not affect the kinetics of activation with respect to ATP but leads to modest increases in the K(m) for tyrosine. Conversely, position Asn- 123 mutants are strongly affected: 160-fold lower k(cat) and 5-fold higher K(m) for the Ala- 1 23; 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 k(cat)/K(m) 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.