title: Lysyl-tRNA synthetase from Bacillus stearothermophilus. Stopped-flow kinetic analysis of enzyme.lysyladenylate formation.
abstract: Amino acid activation reaction of the lysyl-tRNA synthetase [L-lysine:tRNALys ligase (AMP forming); EC 6.1.1.6] from Bacillus stearothermophilus was studied fluorometrically by the stopped-flow method. The addition of L-lysine to the enzyme solution caused quenching of the protein fluorescence and the subsequent addition of ATP restored the quenched fluorescence [Takita et al. (1996) J. Biochem. 119, 680-689; Takita et al. (1997) 121, 244-250]. In the stopped-flow analysis, however, the former fluorescence change (quenching) could not be detected, while the latter change (restoration) was detectable. The L-lysine binding process was suggested to be much faster than the ATP binding process, being completed within the dead-time of the apparatus, ca. 3 ms. The hyperbolic dependence of kapp on the initial ATP concentration suggested that the ATP binding to the enzyme.L-lysine complex followed a two-step mechanism. Two L-lysine analogues that exhibit the qualitatively similar behavior to L-lysine in the fluorometric titration, L-lysine hydroxamate and L-lysine amide, were examined similarly. The two-step process was also suggested for these analogues, and the forward rate constant in the rate-determining step for L-lysine amide (221+/-7 s-1) was significantly larger than those for L-lysine (45.7+/-4.6 s-1) and L-lysine hydroxamate (14. 5+/-1.7 s-1) at pH 8.0, 30 degrees C.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto, 606-8502, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Takita
authors.forename: E
authors.initials: E
authors.lastname: Akita
authors.forename: K
authors.initials: K
authors.lastname: Inouye
authors.forename: B
authors.initials: B
authors.lastname: Tonomura
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D008250:Lysine-tRNA Ligase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D000255:Adenosine Triphosphate; D008250:Lysine-tRNA Ligase