Tryptophanyl-tRNA synthetase from Bacillus subtilis. Characterization and role of hydrophobicity in substrate recognition.

The tryptophanyl-tRNA synthetase from Bacillus subtilis was purified to homogeneity and characterized. It has an alpha 2 subunit structure and a molecular weight of 77,000. Tryptophanyl-tRNA synthetase does not catalyze any significant proofreading. It activates tryptophan as well as the three fluorinated analogues, DL-4-fluoro-, DL-5-fluoro-, or DL-6-fluorotryptophan (4F-, 5F-, and 6F-Trp), in the ATP-pyrophosphate exchange reaction. In the aminoacylation reaction, the fluorotryptophans act as competitive inhibitors of Trp. Their relative activities follow the same order in both reactions: Trp greater than 4F-Trp greater than 6F-Trp greater than 5F-Trp. This order is the inverse of the order of relative hydrophobicities of these compounds, pointing to the importance of hydrophobic interactions in the selective recognition by tryptophanyl-tRNA synthetase among this group of substrates. To define the physical basis of the relative hydrophobicities, the crystallographic structure of 4F-Trp was determined and compared to that of trptophan. Charge distributions calculated for tryptophan and its different fluoroanalogues on the basis of molecular structures were supported by their carbon-13 NMR spectra. Correlations between charge distributions and relative hydrophobicities suggest that the polarity of the C-F bond represents an underlying factor determining the hydrophobicities of 4F-, 5F-, and 6F-Trp, thus relating tryptophanyl-tRNA synthetase selectivity toward tryptophan and its fluoroanalogues directly to their electronic configurations.