Escherichia coli tryptophanyl-tRNA synthetase mutants selected for tryptophan auxotrophy implicate the dimer interface in optimizing amino acid binding.

Tryptophan auxotrophs of Escherichia coli in which mutations were mapped to the trpS locus (encoding tryptophanyl-tRNA synthetase) have been previously isolated. We have investigated the tryptophanyl-tRNA synthetase (TrpRS) purified from six auxotrophic strains for changes in amino acid activation and aminoacylation. Steady-state kinetic analyses show that these mutant TrpRS proteins have increases in the apparent KM for tryptophan, decreases in turnover number, or both, without significant changes in the apparent KM for ATP or tRNA(Trp). The crystal structure of a highly homologous tryptophanyl-tRNA synthetase from Bacillus stearothermophilus in a complex with the cognate aminoacyl adenylate allowed us to place the mutations in a structural context. The mutations in the enzymes are located in the KMSKS loop (M196I), in or near the active site (D112E, P129S, A133E) or far from the active site. The last three mutants (T60R, L91F, G329S) could not be predicted by examination of the protein structure as they line an interface between the C-terminal alpha-helix of one subunit and the Rossmann folds of both subunits, thus affecting a specific region of the dimer interface. These results support a role for dimerization in properly configuring the two active sites of the dimeric enzymes in the Trp/Tyr subclass of class I aminoacyl-tRNA synthetases in order to achieve optimal catalysis.