Abstract
Lysyl-tRNA synthetase from Bacillus stearothermophilus (B.s. LysRS) (EC ) catalyzes aminoacylation of tRNA(Lys) with l-lysine, in which l-lysine was first activated with ATP to yield an enzyme (lysyladenylate complex), and then the lysine molecule was transferred from the complex to tRNA(Lys). B.s. LysRS is a homodimeric enzyme with a subunit that consists of two domains, an N-terminal tRNA anticodon-binding domain (TAB-ND: Ser(1)-Pro(144)) and a C-terminal Class II-specific catalytic domain (CAT-CD: Lys(151)-Lys(493)). CAT-CD alone retained catalytic activity, although at a low level; TAB-ND alone showed no activity. Size exclusion chromatography revealed that CAT-CD exists as a dimer, whereas TAB-ND was a monomer. The formation of a complex consisting of these domains was detected with the guidance of surface plasmon resonance. In accordance with this, the addition of TAB-ND to CAT-CD significantly enhanced both the l-lysine activation and the tRNA aminoacylation reactions. Kinetic analysis showed that deletion of TAB-ND resulted in a significant destabilization of the transition state of CAT-CD in the l-lysine activation reaction but had little effect on the ground state of substrate binding. A significant role of a cross-subunit interaction in the enzyme between TAB-ND and CAT-CD was proposed for the stabilization of the transition state in the l-lysine activation reaction.
Highlights
AaRS1 catalyzes the ligation of an amino acid to the cognate tRNA, generally according to the following (Reaction Scheme 1)
LysRS is a homodimeric enzyme with a subunit that consists of two domains, an N-terminal tRNA anticodon-binding domain (TAB-ND: Ser1-Pro144) and a C-terminal Class IIspecific catalytic domain (CAT-CD: Lys151-Lys493)
It has been suggested that the interface between the anticodon-binding domain and the catalytic domain plays an important role in the tRNA-dependent conformational change in the active site, in GlnRS, which requires the cognate tRNA for the amino acid activation reaction [17], and in MetRS, which does not require it [3, 18]
Summary
AaRS1 catalyzes the ligation of an amino acid to the cognate tRNA, generally according to the following (Reaction Scheme 1),. Lysyl-tRNA Synthetase from B. stearothermophilus the anticodon-binding domain) have been purified separately [7] In this system, it was shown that in both the tRNA-independent amino acid activation and the aminoacylation reactions, the Cterminal non-catalytic domain contributed significantly to the stabilization of the transition state, but not to the ground state of substrate binding. It was shown that in both the tRNA-independent amino acid activation and the aminoacylation reactions, the Cterminal non-catalytic domain contributed significantly to the stabilization of the transition state, but not to the ground state of substrate binding In this case, because the catalytic domain of HisRS was purified as monomer, it was not clear whether the observed destabilization of the transition state upon deletion of the non-catalytic domain was because of the dissociation of the catalytic domain into a monomer or because of the loss of the interaction between the catalytic domain and the non-catalytic domain. The aim of this study was to elucidate the role of the interaction between the catalytic domain and the anticodon-binding domain in the tRNA-independent L-lysine activation reaction, and to elucidate the mechanism of the transition state stabilization in the enzymatic reaction
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