Lysyl-tRNA 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 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.
Takita
T
T
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University.
Ohkubo
Y
Y
Shima
H
H
Muto
T
T
Shimizu
N
N
Sukata
T
T
Ito
H
H
Saito
Y
Y
Inouye
K
K
Hiromi
K
K
Tonomura
B
B
D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism*; D008250:Lysine-tRNA Ligase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D000255:Adenosine Triphosphate; D008250:Lysine-tRNA Ligase; D008239:Lysine