Lysyl-tRNA synthetase [L-lysine:tRNA(Lys)ligase (AMP forming); EC 6.1.1.6] was purified from Bacillus stearothermophilus NCA1503 approximately 1,100-fold to homogeneity in PAGE. The enzyme is a homodimer of M(r) 57,700 x 2. The molar absorption coefficient, epsilon, at 280 nm is 71,600 M-1.cm-1 at pH8.0. Enzyme activity in the tRNA aminoacylation reaction and the ATP-PPi exchange reaction increases up to 50 degrees C at pH 8.0, but is lost completely at 70 degrees C. The pH-optima of the two reactions are 8.3 at 37 degrees C. In the tRNA aminoacylation reaction, the Km values for L-lysine and ATP are 16.4 and 23.2 muM, respectively, and in the ATP-PPi exchange reaction, the Km values for L-lysine and ATP are 23.6 and 65.1 muM, respectively at 37 degrees C, pH 8.0. Interaction of either L-lysine or ATP with the enzyme has been investigated by using as a probe the ligand-induced quenching of protein fluorescence and by equilibrium dialysis. These static analyses, as well as the kinetic analysis of the L-lysine dependent ATP-PPi exchange reaction indicate that the binding mode of L-lysine and ATP to the enzyme is sequential ordered (L-lysine first). The interaction of lysine analogues with the enzyme has also been investigated.