Lysyl-tRNA synthetase from Bacillus stearothermophilus. Formation and isolation of an enzyme-lysyladenylate complex and its analogue.
The formation of an enzyme.lysyladenylate complex was studied with a highly purified lysyl-tRNA synthetase [L-lysine:tRNALYS 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 microM, respectively, at pH 8.0, 30 degrees C, by fluorometric measurement. The isolated enzyme.lysyladenylate complex was relatively stable with a rate constant of decomposition of 1.7 x 10(-5) s-1 at pH 8.5 and 0 degree C. The rate constant of transfer of L-lysine from the complex to Escherichia coli tRNA was 1.2 x 10(-2) S-1 at pH 8.5 and 0 degree C. The effects of replacing L-lysine by several analogues on the complex formation were examined. L-Lysine hydroxamate, a strong inhibitor of the L-lysine dependent ATP-PPi exchange reaction, produced 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.
Takita
T
T
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University.
Hashimoto
S
S
Ohkubo
Y
Y
Muto
T
T
Shimizu
N
N
Sukata
T
T
Inouye
K
K
Hiromi
K
K
Tonomura
B
B
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D004492:Edetic Acid / Q000494:pharmacology; D005374:Filtration; D005470:Fluorometry; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism*; D008250:Lysine-tRNA Ligase / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D011485:Protein Binding
D002614:Chelating Agents; D046911:Macromolecular Substances; C039763:lysine hydroxamate; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D008250:Lysine-tRNA Ligase; D008239:Lysine