ESCHERICHIA-COLI TRYPTOPHANYL-TRANSFER-RNA SYNTHETASE MUTANTS SELECTED FOR TRYPTOPHAN AUXOTROPHY IMPLICATE THE DIMER INTERFACE IN OPTIMIZING AMINO-ACID BINDING

Tryptophan auxotrophs of Escherichia coli in which mutations were mapp ed to the trpS locus (encoding tryptophanyl-tRNa synthetase) have been previously isolated. We have investigated the tryptophanyl-tRNA synth etase (TrpRS) purified from six auxotrophic strains for changes in ami no acid activation and aminoacylation. Steady-state kinetic analyses s how that these mutant TrpRS proteins have increases in the apparent K- M for tryptophan, decreases in turnover number, or both, without signi ficant changes in the apparent K-M for ATP or tRNA(Trp). The crystal s tructure of a highly homologous tryptophanyl-tRNA synthetase from Baci llus stearothermophilus in a complex with the cognate aminoacyl adenyl ate allowed us to place the mutations in a structural context, The mut ations in the enzymes are located in the KMSKS loop (M196I), in or nea r the active site (D112E, P129S, A133E) or far from the active site, T he last three mutants (T60R, L91F, G329S) could not be predicted by ex amination of the protein structure as they line an interface between t he C-terminal alpha-helix of one subunit and the Rossmann folds of bot h subunits, thus affecting a specific region of the dimer interface. T hese results support a role for dimerization in properly configuring t he two active sites of the dimeric enzymes in the Trp/Tyr subclass of class I aminoacyl-tRNA synthetases in order to achieve optimal catalys is.