Lysyl-tRNA Synthetase from Bacillus stearothermophilus. Purification, and Fluorometric and Kinetic Analysis of the Binding of Substrates, L-Lysine and ATP

抄録

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, 700×2. The molar absorption coefficient, ε, at 280nm is 71, 600M^-1•cm^-1 at pH 8.0. Enzyme activity in the tRNA aminoacylation reaction and the ATP-PP₁ exchange reaction increases up to 50°C at pH 8.0, but is lost completely at 70°C. The pH-optima of the two reactions are 8.3 at 37°C. In the tRNA aminoacylation reaction, the K_m values for L-lysine and ATP are 16.4 and 23.2μM, respectively, and in the ATP-PP₁ exchange reaction, the K_m values for L-lysine and ATP are 23.6 and 65.1μM, respectively at 37°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-PP₁ 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.