caa a22 4500 397557434 CHVBK 20180308164806.0 cr unu---uuuuu 161202e199604 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021296 doi (NATIONALLICENCE)oxford-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 [Elektronische Daten] [Teisuke Takita, Yuji Ohkubo, Hideaki Shima, Takanori Muto, Naofumi Shimizu, Tokuo Sukata, Hiroshi Ito, Yukiko Saito, Kuniyo Inouye, Keitaro Hiromi, Ben'ichiro Tonomura] 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. © by the Journal of Biochemistry Regular Papers nationallicence aminoacyl-tRNA synthetase nationallicence fluorescence titration nationallicence lysyl-tRNA synthetase nationallicence protein fluorescence nationallicence substrate binding order nationallicence Takita Teisuke Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut Ohkubo Yuji Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut Shima Hideaki Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut Muto Takanori Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut Shimizu Naofumi Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut Sukata Tokuo Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut Ito Hiroshi Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut Saito Yukiko Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut Inouye Kuniyo Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut Hiromi Keitaro Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut Tonomura Ben'ichiro Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut The Journal of Biochemistry Oxford University Press 119/4(1996-04), 680-689 0021-924X 119:4<680 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021296 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021296 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Takita Teisuke Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut NATIONALLICENCE 700 1- Ohkubo Yuji Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut NATIONALLICENCE 700 1- Shima Hideaki Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut NATIONALLICENCE 700 1- Muto Takanori Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut NATIONALLICENCE 700 1- Shimizu Naofumi Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut NATIONALLICENCE 700 1- Sukata Tokuo Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut NATIONALLICENCE 700 1- Ito Hiroshi Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut NATIONALLICENCE 700 1- Saito Yukiko Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut NATIONALLICENCE 700 1- Inouye Kuniyo Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut NATIONALLICENCE 700 1- Hiromi Keitaro Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut NATIONALLICENCE 700 1- Tonomura Ben'ichiro Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-01 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/4(1996-04), 680-689 0021-924X 119:4<680 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford