Naslov
Specificity of leucyl-tRNA synthetase’s editing domain is determined by the chemical step of proofreading

Autori
Dulić, Morana ; Palencia, Andres ; Cvetešić, Nevena ; Cusack, Stephen ; Gruić-Sovulj, Ita

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of abstracts, FEBS3+ Meeting "Molecules of Life" / Janko Kos, Nataša Poklar Ulrih - Ljubljana : Slovenian Biochemical Society, 2015, 106-106

ISBN
978-961-93879-1-7

Skup
FEBS3+ Meeting "Molecules of Life"

Mjesto i datum
Portorož, Slovenija, 16.09.2015. - 19.09.2015

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Nije recenziran

Ključne riječi
Leucyl-tRNA synthetase; deacylation; editing; specificity

Sažetak
Leucyl-tRNA synthetase (LeuRS) covalently attaches leucine to tRNALeu in an ATP- dependent manner. LeuRS also catalyzes reactions with noncognate norvaline, a noncanonical amino acid that accumulates under microaerobic conditions. To counter this major threat for the accuracy of leucylation, norvaline is eliminated from protein synthesis by deacylation of mischarged tRNA at a separate LeuRS editing domain. To prevent futile ATP consumption, the editing domain efficiently discriminates against cognate Leu-tRNALeu. Although the general perception is that the editing domain operates as a fine sieve that prevents binding of the cognate substrate, the intriguing question remains whether specificity is exercised predominately through the ground state binding or catalysis. The contribution of ground state binding was examined by measuring the interactions of the isolated LeuRS editing domain from Escherichia coli and the non- hydrolysable analogues of the 3’ end of aminoacylated tRNA (Nva2AA and Leu2AA) using isothermal titration calorimetry. The dissociation constant for Leu2AA was only 10- fold higher than for Nva2AA, indicating a modest contribution of binding to specificity. Moreover, no significant difference in binding affinity of Nva-tRNALeu or Leu-tRNALeu to deacylation-defective LeuRS was observed using microscale thermophoresis. This is likely due to the large interacting surface of the tRNA with LeuRS, which masks the difference in binding of cognate and noncognate amino acid to the editing domain. Next, we compared deacylation of Nva-tRNALeu and Leu-tRNALeu by LeuRS under single turnover conditions. We show that the cognate aa-tRNA is discriminated at the chemical step by 103-fold. We also confirm the lack of significant differences between the Kds for Nva-tRNALeu and Leu-tRNALeu. Our results thus demonstrate that substrate specificity in deacylation is predominately governed by the complementarity established at the transition state. The T252A substitution was previously shown to undermine the enzyme’s specificity by enabling hydrolysis of Leu- tRNALeu. Remarkably, our thermodynamic and kinetic analyses revealed that this substitution does not affect Kd values for Leu2AA or Leu-tRNALeu. Yet, the deacylation rate constant was 500-fold increased as compared to the wild-type enzyme. It thus appears that the major specificity determinant of the editing domain operates by mispositioning of the catalytic residues to prevent hydrolysis of the cognate product.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA