Naslov
Amino acid specificity of the Escherichia coli leucyl-tRNA synthetase editing domain

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

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

Izvornik
FEBS Journal Special Issue: 40th FEBS Congress, The Biochemical Basis of Life, Berlin, Germany, July 4-9, 2015 / - , 2015, 87-88

Skup
40th FEBS Congress, The Biochemical Basis of Life

Mjesto i datum
Berlin, Njemačka, 04.07.2015. - 09.07.2015

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

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

Sažetak
Leucyl-tRNA synthetase (LeuRS) covalently attaches leucine to cognate tRNA in an ATP- dependent manner. This enzyme also catalyses reactions with norvaline ; a noncanonical amino acid that accumulates in Escherichia coli under microaerobic conditions and presents major threat for accuracy of Leu-tRNALeu synthesis. Norvaline is eliminated from protein synthesis by deacylation of Nva-tRNALeu in the LeuRS editing domain. To prevent futile ATP consumption, the editing domain has evolved mechanisms to efficiently discriminate against cognate aminoacyl-tRNA. Both, binding and catalysis may contribute to specificity. It was shown that LeuRS discriminates against Leu- tRNALeu with 103-fold specificity that arises from decreased rate of deacylation in the editing domain. To determine contribution of ground-state binding in specificity, we examined interactions between the LeuRS editing site 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, challenging a general perception that editing domain operates as a fine sieve that prevents binding of the cognate substrate. To explore specificity determinants of the editing site, we introduced T252A substitution that enables Leu-tRNALeu hydrolysis. Interestingly, this substitution did not increase the affinity for Leu2AA, implying that distinct determinants govern discrimination against Leu-tRNALeu at the binding and chemical steps. The full length Nva-tRNALeu and Leu-tRNALeu bind to deacylation-defective LeuRS with similar affinity, as measured by microscale thermophoresis. This further supports the notion that the substrate specificity in deacylation is preferentially established at the transition rather than at the ground-state.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA