engleski

Interplay between synthetic and editing pathways in class I aminoacyl-tRNA synthetases

Extensive steady-state and transient kinetics were used in analysis of synthetic and editing pathways in class I isoleucyl- valyl- and leucyl-tRNA synthetases from Escherichia coli (IleRS, ValRS, LeuRS, respectively). We find that kinetic partitioning of the aminoacyl-adenylate between transfer and hydrolysis in the synthetic site dictates the respective use of pre- versus post- transfer pathways [1]. In ValRS and LeuRS, rapid transfer of threonine and norvaline, respectively, ensures prevalence of the post-transfer pathway. In contrast, in IleRS, tRNA-dependent hydrolytic clearance of valyl-adenylate within the synthetic site competes well with a slow transfer step, making both pre- and post-transfer pathways necessary. We also found that kinetic partitioning between synthetic and editing pathways occurs at both pre- and post-transfer steps [2]. Fast hydrolysis of misacylated tRNAs within the CP1- editing site efficiently competes with their release from the enzyme, while slow deacylation of cognate Leu-tRNALeu ensures that product dissociation predominates. Further, the LeuRS editing site exhibits substantial specificity among aminoacyl-tRNA substrates, and the editing rate is limited by product release. Finally, fundamental differences between two of the primary gatekeepers of cellular fidelity were recognized. DNA-polymerases feature a highly specific synthetic site where cognate and non-cognate substrates are distinguished by binding and reaction kinetics, and its editing site is less specific. In contrast, editing aaRSs predominately use a highly specific editing site to eliminate non-cognate substrates. Dulic M, Cvetesic N, Perona JJ, Gruic-Sovulj I. (2010) J Biol Chem. 285, 23799-809. Cvetesic N, Perona JJ, Gruic-Sovulj I. (2012) J Biol Chem. 287, 25381-94.

leucyl-tRNA synthetase; isoleucyl-tRNA synthetase; hydrolytic editing; norvaline

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