Interactions of several amino acids and nucleotides with valyl-tRNA synthetase [EC 6.1.1.9] (VRS) from Bacillus stearothermophilus were investigated using as a probe the ligand-induced quenching of protein fluorescence (lambda ex = 295 nm, lambda em = 340 nm) of VRS. L-Valine, L-threonine, L-isoleucine, L-glutamic acid, L-leucine, and D-valine caused fluorescence quenching. Among them, L-threonine had a Kd value comparable to that for the cognate substrate, L-valine, but the other amino acids were bound more weakly as estimated by the fluorescence titration method. L-Alanine, L-histidine, and L-serine did not cause any fluorescence change. Among the nucleotides tested (ATP, ADP, AMP, GTP, ITP, CTP, and UTP), only ATP caused the fluorescence change. In the presence of an excess amount of ATP, only L-valine and L-threonine, among the tested amino acids, induced the fluorescence quenching, and the binding of L-valine was greatly favored under this condition. This is consistent with the results of the ATP-PP1 exchange reaction by VRS, in which only L-valine and L-threonine, of these 9 amino acids tested, could serve as substrates, and the Km value for L-valine was much smaller than that for L-threonine. Thus the binding of ATP to VRS enhances the substrate specificity of VRS towards amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)