Biopolymers and cell. 2010; 26 (6): 478 - 485
Functional role of C-terminal domain of Thermus thermophilus leucyl-tRNA synthetase
O. I. Gudzera, A. D. Yaremchuk, M. A. Tukalo
Institute of Molecular Biology and Genetics NAS of Ukraine, 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
Aim. To study a role of C-terminal domain of T. thermophilus leucyl-tRNA synthetase (LeuRSTT) in the reactions of aminoacylation and editing. Methods. A mutant of LeuRSTT without C- terminal domain (ΔС) was obtained by the method of mutagenesis. The kinetic constants in aminoacylation reaction catalyzed by LeuRS and its mutant (ΔС) were determined by the methods of equilibrium enzyme kinetics. To evaluate the contribution of C-terminal domain to interaction of the enzyme with tRNALeu, Kd of a complex between tRNA and LeuRSTT and its mutant ΔС was determined by fluorescence titration. Results. The C-terminal domain is shown to play a significant role in the aminoacylation and editing reactions of LeuRSTT and not essential for the activity in the reaction of amino acid activation. The kinetic parameters of aminoacylation of tRNALeu and tRNATyr by LeuRS and ΔС mutant were also determined, their analysis suggests that the C-domain is not critical for the manifestation of specificity of the enzyme in the recognition of homologous RNAs. At the same time a significant influence of the C-terminal domain on the value of catalytic constant was shown. At the domain deletion the kcat value is lower by 152-fold. Conclusion. The C-terminal domain of LeuRSTT is evolutionarily acquired to enhance the rate of catalysis in the aminoacylation and editing reactions, and makes no significant contribution to the specificity of the enzyme in the recognition of tRNA.
Keywords: leucyl-tRNA synthetase, tRNALeu, editing.