Kinetic Origin of Substrate Specificity in Post-Transfer Editing by Leucyl-tRNA Synthetase
| Autor: | Ita Gruić-Sovulj, Stephen Cusack, Branimir Bertoša, Andrés Palencia, Igor Zivkovic, Morana Dulic, Nevena Cvetesic |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Stereochemistry Acylation Aminoacylation RNA Transfer Amino Acyl Biology Substrate Specificity Amino Acyl-tRNA Synthetases 03 medical and health sciences chemistry.chemical_compound Structural Biology Escherichia coli Amino Acids Molecular Biology chemistry.chemical_classification Binding Sites Aminoacyl tRNA synthetase Hydrolysis Leucyl-tRNA synthetase Translation (biology) Amino acid Kinetics 030104 developmental biology chemistry Biochemistry Transfer RNA Leucine-tRNA Ligase aminoacyl-tRNA synthetases proofreading catalytic RNA substrate-assisted catalysis norvaline Leucine Norvaline |
| Zdroj: | Journal of Molecular Biology. 430:1-16 |
| ISSN: | 0022-2836 |
| DOI: | 10.1016/j.jmb.2017.10.024 |
| Popis: | The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNALeu and their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNALeu differed by about 104-fold. We further established the critical role for the A76 3′-OH group of the tRNALeu in post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNALeu hydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection. |
| Databáze: | OpenAIRE |
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