engleski

Distinct origin of amino acid substrate specificity by tRNA synthetase synthetic and editing sites

Aminoacyl-tRNA synthetases (aaRS) catalyze ATP- dependent covalent coupling of cognate amino acids and tRNAs for ribosomal protein synthesis. The inability of some aaRSs to discriminate efficiently against structurally similar near- cognate amino acids solely during the binding or catalytic steps of the aminoacylation reaction prompted the evolution of hydrolytic proofreading in these enzymes. The prominent error-correction step is hydrolysis of the incorrect final product, misacylated tRNA, within a dedicated protein domain (post-transfer editing). Also, the near- cognate aminoacyl-adenylate intermediate may be proofread within the synthetic site (pre- transfer editing). Leucyl-tRNA synthetase (LeuRS) provides a cellular pool of Leu-tRNALeu. The enzyme from Escherichia coli utilizes vigorous editing reaction that hydrolyses misacylated tRNAs within the LeuRS domain dedicated for post-transfer editing. We have recently shown that this proofreading activity is essential under microaerobic growth conditions in which accumulated non-proteinogenic amino acid norvaline may jeopardize the accuracy of Leu- tRNALeu synthesis [1]. Using quantitative shotgun proteomics we confirmed incorporation of norvaline at 10% of all leucine sites in the proteome of the E. coli strain deprived of LeuRS editing under oxygen-limiting conditions. We further revealed that the commonly held view in which LeuRS frequently misactivates isoleucine in vitro is mistaken because it is based on measurements with impure isoleucine samples that contain traces of leucine. Extensive kinetic and thermodynamic analyses of the synthetic and editing pathways indicated that editing aaRSs use fundamentally different mechanisms to strengthen specificity in the synthetic and editing sites. We found that near-cognate amino acids are generally not discriminated at the chemical steps of aminoacylation [2, 3]. This distinguishes aaRSs from some other proofreading enzymes, like DNA polymerases. The synthetic sites of LeuRS and the closely related isoleucyl-tRNA synthetases (IleRS) yet contribute to specificity of aminoacylation by promoting a 100-fold difference in the Km values for cognate and near-cognate amino acids. In contrast, the committed post- transfer editing sites in these enzymes exclude the corresponding cognate aminoacyl-tRNA, with 103-fold specificity that arises from decreased rate of deacylation. The Kd values describing binding of the cognate and near-cognate post- transfer editing substrates to LeuRS were similar, challenging a general perception in which the editing domain operates as a steric sieve. To support this notion we showed that the major specificity determinant of the LeuRS editing domain operates by mispositioning of the catalytic residues to prevent hydrolysis of the cognate product. [1] Nevena Cvetesic, Andrés Palencia, Ivan Halasz, Stephen Cusack, Ita Gruic- Sovulj (2014) EMBO J. 33, 1639–1653. [2] Cvetesic N, Perona JJ, Gruic-Sovulj I. (2012) J Biol Chem. 287, 25381-94. [3] Dulic M, Cvetesic N, Perona JJ, Gruic-Sovulj I. (2010) J Biol Chem. 285, 23799- 809.

aminoacyl-tRNA synthetases, editing, norvaline, leucyl-tRNA synthetase

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