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- Title
Lysyl-tRNA Synthetase from Bacillus stearothermophilus. Purification, and Fluorometric and Kinetic Analysis of the Binding of Substrates, L-Lysine and ATP1.
- Authors
Takita, Teisuke; Ohkubo, Yuji; Shima, Hideaki; Muto, Takanori; Shimizu, Naofumi; Sukata, Tokuo; Ito, Hiroshi; Saito, Yukiko; Inouye, Kuniyo; Hiromi, Keitaro; Tonomura, Ben'ichiro
- Abstract
Lysyl-tRNA synthetase [L-lysine: tRNALys; 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 Mr 57,700x2. The molar absorption coefficient, €, at 280 nm is 71,600 M−1·cm−1 at pH 8.0. Enzyme activity in the tRNA aminoacylation reaction and the ATP-PP1 exchange reaction increases up to 50°C at pH 8.0, but is lost completely at 70°C. The pH-optima of the two reactions are 8.3 at 37°C. In the tRNA aminoacylation reaction, the Km values for L-lysine and ATP are 16.4 and 23.2 μM, respectively, and in the ATP-PP1, exchange reaction, the Km values for L-lysine and ATP are 23.6 and 65.1 μM, respectively at 37°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-PP1, 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.
- Subjects
HIGH-lysine diet; AMINO acids; LYSINE; ENZYMES; LYSYL-tRNA synthetase
- Publication
Journal of Biochemistry, 1996, Vol 119, Issue 4, p680
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/oxfordjournals.jbchem.a021296