J. Biochem, 1996, Vol. 119, No. 4 680-689
© 1996 Japanese Biochemical Society
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Lysyl-tRNA Synthetase from Bacillus stearothermophilus. Purification, and Fluorometric and Kinetic Analysis of the Binding of Substrates, L-Lysine and ATP1
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University Kitashirakawa, Kyoto 606-01
2 To whom correspondence should be addressed.
Lysyl-tRNA synthetase [L-lysine: tRNALys; ligase (AMP forming); EC 6.1.1.6
[EC]
] 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.
1 This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and a grant from the Japan Foundation of Applied Enzymology.
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