1989 Fiscal Year Final Research Report Summary
Studies on the reaction mechanism of aminoacyl-tRNA synthetases.
| Project/Area Number |
62560081
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
応用生物化学・栄養化学
|
|---|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
TONOMURA Ben'ichiro Kyoto University, Faculty of Agriculture, Professor, 農学部, 教授 (20026545)
|
|---|
| Project Period (FY) |
1987 – 1989
|
| Keywords | Aminoacyl-tRNA synthetase / Valyl-tRNA synthetase / Lysyl-tRNA synthetase / Specific binding of enzyme and substrate / Binding order of two substrates and enzyme / Fluorometric titration / Equilibrium dialysis / 3'-o-Anthrailoyl ATP |
|---|
| Research Abstract |
1. A valyl-tRNA synthetase (EC 6.1.1.9)(VRS) was isolated from Bacillus stearothermophilus NCA 1503 to electrophoretical homogeneity. The static and kinetic aspects of the interaction between VRS and a fluorescent analogue of ATP, 3'-O-anthraniloyl ATP (Ant-ATP), was studied. It was found for the first time that Ant-ATP was a substrate of VRS in the formation of valyl-tRNA. The fluorescence of Ant-ATP increased on the binding of VRS, and the equilibrium dissociation constant (Kd) was determined by titration with this fluorescence change as probe. Fast kinetic studies on the interaction of VRS and ANT-ATP were made with micro-stopped-flow apparatus by using the fluorescence change as probe. The kinetic feature was consistent with a two-step mechanism, and the kinetic parameters relevant to the mechanism were estimated.
2. A lysyl-tRNA syntletase (EC 6.1.1.6) (LRS) was isolated from Bacillus stearothermophilus NCA 1503 to electrophoretical homogeneity. LRS consists of 2 identical subunits of 60 kD, Mw being 120,000. Steady state kinetic parameters for the amino acid activation and the tRNA amino-acylation reactions were determined. The results of equilibrium dialysis, fluorometric titration with the intrinsic protein fluorescence as probe, and steady-state kinetic analysis in the presence of inhibitors suggest, for the amino acid activation reaction, that (1) L-lysine is bound to LRS first and the binding of ATP follows in a sequential mechanism; (2) out of the two identical active sites per dimer only one shows the catalytic activity at one time; (3) The binding of ATP to LRS enhances the degree of recognition by LRS towards the side chain structure of amino acid substrates.
|
