Ancient adaptation of the active site of tryptophanyl-tRNA synthetase for tryptophan binding

The amino acid binding: domains of the tryptophanyl (TrpRS)- and tyrosyl-tR NA synthetases (TyrRS) of Bacillus stearothermophilus are highly homologous . These similarities suggest that conserved residues in TrpRS may be respon sible for both determining tryptophan recognition and discrimination agains t tyrosine. This was investigated by the systematic mutation of TrpRS resid ues based upon the identity of homologous positions in TyrRS. Of the four r esidues which interact directly with the aromatic side chain of tryptophan (Phe5, Met129, Asp132, and Val141) replacements of Asp 132 led to significa nt changes in the catalytic efficiency of Trp aminoacylation (200-1250-fold reduction in k(cat)/K-M) and substitution of Val141 by the larger Glu side chain reduced k(cat)/K-M by 300-fold. Mutation of Pro127, which determines the position of active-site residues, did not significantly effect Trp bin ding. Of the mutants tested, D132N TrpRS also showed a significant reductio n in discrimination against Tyr, with Tyr acting as a competitive inhibitor but not a substrate. The analogous residue in B. stearothermophilus TyrRS (Asp176) has also been implicated as a determinant of amino acid specificit y in earlier studies [de Prat Gay, G., Duckworth, H. W., and Fersht, A. R. (1993) FEES Lett. 318, 167-171]. This striking similarity in the function o f a highly conserved residue found in both TrpRS and TyrRS provides mechani stic support for a common origin of the two enzymes.