LYSYL-TRANSFER-RNA SYNTHETASE FROM BACILLUS-STEAROTHERMOPHILUS - FORMATION AND ISOLATION OF AN ENZYME-CENTER-DOT-LYSYLADENYLATE COMPLEX ANDITS ANALOG

The formation of an enzyme . lysyladenylate complex was studied with a highly purified lysyl-tRNA synthetase [L-lysine:tRNA(Lys) ligase (AMP -forming); EC 6.1.1.6] from Bacillus stearothermophilus. The apparent dissociation equilibrium constants of the enzyme with L-lysine and ATP in the process of the complex formation were estimated to be 50.9 and 15.5 mu M, respectively, at pH 8.0, 30 degrees C, by fluorometric mea surement. The isolated enzyme-lysyladenylate complex was relatively st able with a rate constant of decomposition of 1.7 x 10(-5)s(-1) at pH 8.5 and 0 degrees C. The rate constant of transfer of L-lysine from th e complex to Escherichia coli tRNA was 1.2 x 10(-2)s(-1) at pH 8.5 and 0 degrees C. The effects of replacing L-lysine by several analogues o n the complex formation were examined. L-Lysine hydroxamate, a strong inhibitor of the L-lysine dependent ATP-PP1 exchange reaction, produce d a stable complex with the enzyme and ATP, enzyme . lysinehydroxamate -AMP probably being formed. The binding stoichiometry of the assumed L -lysinehydroxamate-AMP per mol of the dimer enzyme was 1:1.