LYSYL-TRANSFER-RNA SYNTHETASE FROM BACILLUS-STEAROTHERMOPHILUS - PURIFICATION, AND FLUOROMETRIC AND KINETIC-ANALYSIS OF THE BINDING OF SUBSTRATES, L-LYSINE AND ATP

Lysyl-tRNA synthetase [L-lysine:tRNA(Lys) ligase (AMP forming); EC 6.1 .1.6] was purified from Bacillus stearothermophilus NCA1503 approximat ely 1,100-fold to homogeneity in PAGE. The enzyme is a homodimer of M( r) 57,700X2. The molar absorption coefficient, epsilon, at 280 nm is 7 1,600 M(-1) . cm(-1) at pH 8.0. Enzyme activity in the tRNA aminoacyla tion reaction and the ATP-PPi exchange reaction increases up to 50 deg rees C at pH 8.0, but is lost completely at 70 degrees C. The pH-optim a of the two reactions are 8.3 at 37 degrees C. In the tRNA aminoacyla tion reaction, the K-m values for L-lysine and ATP are 16.4 and 23.2 m u M, respectively, and in the ATP-PPi exchange reaction, the K-m value s for L-lysine and ATP are 23.6 and 65.1 mu M, respectively at 37 degr ees C, pH 8.0. Interaction of either L-lysine or ATP with the enzyme h as been investigated by using as a probe the ligand-induced quenching of protein fluorescence and by equilibrium dialysis, These static anal yses, as well as the kinetic analysis of the L-lysine dependent ATP-PP i 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.