Journal Article

Lysyl-tRNA Synthetase from Bacillus stearothermophilus. Purification, and Fluorometric and Kinetic Analysis of the Binding of Substrates, L-Lysine and ATP

Teisuke Takita, Yuji Ohkubo, Hideaki Shima, Takanori Muto, Naofumi Shimizu, Tokuo Sukata, Hiroshi Ito, Yukiko Saito, Kuniyo Inouye, Keitaro Hiromi and Ben'ichiro Tonomura

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 119, issue 4, pages 680-689
Published in print April 1996 | ISSN: 0021-924X
Published online April 1996 | e-ISSN: 1756-2651 | DOI: https://dx.doi.org/10.1093/oxfordjournals.jbchem.a021296
Lysyl-tRNA Synthetase from Bacillus stearothermophilus. Purification, and Fluorometric and Kinetic Analysis of the Binding of Substrates, L-Lysine and ATP

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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.

Keywords: aminoacyl-tRNA synthetase; fluorescence titration; lysyl-tRNA synthetase; protein fluorescence; substrate binding order

Journal Article.  0 words. 

Subjects: Biochemistry

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