Abstract
A fluorescent analogue of ATP, 3′-O-antraniloyl-ATP (Ant-ATP), was found to serve as a substrate of valyl-tRNAVal synthetase (l-valine:tRNAVal ligase (AMP-forming), EC 6.1.1.9) from Bacillus stearothermophilus NCA 1503 in the tRNA aminoacylation reaction in place of ATP; Km and kcat at pH 7.5 and 30°C were 440 μM and 0.58 s−1, respectively, whereas those for ATP were 34 μM and 2.9 s−1, respectively. The fluorescence of Ant-ATP (λem = 428 nm) changed significantly on the binding with the enzyme; Kd of the enzyme and Ant-ATP was determined by fluorometric titration to be 290 μM at pH 7.5, 30°C. Fast kinetic studies on the interaction of the enzyme and Ant-ATP were made with a micro-stopped-flow apparatus by using the fluorescence change as probe. The kinetic feature was consistent with a two-step mechanism in which a fast bimolecular process is followed by a unimolecular isomerization process. Kinetic parameters relevant to the mechanism were estimated.
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