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

Abstract

Lysyl-tRNA synthetase [L-lysine:tRNA(Lys)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 M(r) 57,700 x 2. The molar absorption coefficient, epsilon, at 280 nm is 71,600 M-1.cm-1 at pH8.0. Enzyme activity in the tRNA aminoacylation reaction and the ATP-PPi exchange reaction increases up to 50 degrees C at pH 8.0, but is lost completely at 70 degrees C. The pH-optima of the two reactions are 8.3 at 37 degrees C. In the tRNA aminoacylation reaction, the Km values for L-lysine and ATP are 16.4 and 23.2 muM, respectively, and in the ATP-PPi exchange reaction, the Km values for L-lysine and ATP are 23.6 and 65.1 muM, respectively at 37 degrees 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-PPi 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.

Divergence in Noncognate Amino Acid Recognition between Class I and Class II Lysyl-tRNA Synthetases

“…Following 30 min of incubation at 37°C, an aliquot was removed and treated with RNase P1. The liberated [␣- 32 …”

Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

“…Binding order appears to be enzyme specific, as it is not conserved even within the LysRS [28], however a recent series of papers has elucidated the mechanism of the Bacillus stearothermophilus lysyl-tRNA synthetase [29-31]. As in our experiments, it was observed that lysine binding causes a significant change in fluorescence whilst ATP only causes a small change that was not quantifiable.…”

“…It has already been established that AMPPCP is a good analogue of the bound ATP hence binding affinities are expected to be in the same range. Supporting this assumption, a dissociation constant corresponding to a Δ G binding of ca -7 kcal/mol for ATP association to B. stearothermophilus LysRS has been reported [29]. …”

Transition State Stabilization by the N-terminal Anticodon-binding Domain of Lysyl-tRNA Synthetase

“…The k d value was obtained as the linear slope of the ln(R 0 /R n ) versus (t n -t 0 ) plot. ATP-PPi Exchange Reaction-The L-lysine activation reaction was measured by using the L-lysine-dependent ATP-PPi exchange reaction at pH 8.0, 37°C, as reported previously (22). The standard reaction mixture contained in 0.5 ml: 100 mM Tris-HCl buffer (pH 8.0), 10 mM MgCl 2 , ATP, L-lysine, and PPi (9.1 mCi/mmol).…”

“…Aminoacylation Reaction-The tRNA aminoacylation reaction was measured at pH 8.0, 37°C, as reported previously (22). The standard reaction mixture contained in 0.…”