Naslov
NEGATIVE CATALYSIS BY ISOLEUCYL-tRNA SYNTHETASE: HOW BROAD SUBSTRATE SELECTIVITY DOES NOT PROMOTE FUTILE EDITING CYCLES

Autori
Živković, Igor ; Maršavelski, Aleksandra ; Gruić- Sovulj, Ita

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

Izvornik
Book of Abstracts of the Congress of the Croatian Society of Biochemistry and Molecular Biology HDBMB22: From Science to Knowledge / Dulić, Morana ; Sinčić, Nino ; Vrhovac Madunić, Ivana - Zagreb : Hrvatsko Društvo za Biotehnologiju, 2022, 164-164

Skup
Congress of the Croatian Society of Biochemistry and Molecular Biology: From Science to Knowledge (HDBMB22)

Mjesto i datum
Brela, Hrvatska, 28.09.2022. - 01.10.2022

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
Aminoacyl-tRNA synthetases, post-transfer editing, negative catalysis, amino acid specificity

Sažetak
Aminoacyl-tRNA synthetases (AARS) activate amino acids and transfer them to cognate tRNAs. Some AARS cannot establish the required specificity in the initial amino acid recognition at the synthetic site and thus may erroneously activate non-cognate amino acids and transfer them onto tRNA. To ensure the faithful protein biosynthesis, those aminoacyl-tRNA synthetases evolved a separate editing domain aimed to hydrolyse erroneously aminoacylated tRNAs (post-transfer editing). To understand what shaped the selectivity of the editing site we used Escherichia coli isoleucyl-tRNA synthetase as a model enzyme. We investigated the kinetics of synthetic and editing reactions of IleRS using a wide range of amino acids, from proteinogenic to synthetic: Val, Nva, Leu, Thr, Met, Ser, Ala, Abu (α-aminobutyrate), Nle (norleucine), F2Abu (γ, γ- difluoro-α-aminobutyrate) and F3Abu (γ, γ, γ- trifluoro-α-aminobutyrate). Among these, only Val and Nva were poorly discriminated (well activated and transferred to the tRNA) and thus can be considered as a threat to the fidelity of translation. To our surprise, tRNAs misaminoacylated with all tested amino acids were rapidly hydrolysed at the editing domain, showing that amino acid’s physicochemical features or how well they are discriminated at the synthetic site and consequently whether or not they can jeopardize the translational fidelity, does not make a significant difference. Only the hydrolysis of cognate Ile-tRNAIle (misediting) was slow, suggesting that the need to keep the cognate product out of editing strongly shaped the specificity of the editing domain. Detailed kinetic analysis revealed that isoleucyl-tRNA synthetase employs Thr246 and His333 for specific destabilization of Ile-tRNAIle hydrolysis (i.e. negative catalysis). Such design enabled isoleucyl-tRNA synthetase to have broad substrate acceptance at the editing site whilst maintaining a high specificity towards preventing the futile post-transfer editing cycles. This was the first time such broad substrate specificity, paired with negative catalysis, was observed for an AARS, and as such, it marks a new moment in the understanding of these vital and ancient enzymes. Živković I., et al. ; Negative catalysis by the editing domain of class I aminoacyl-tRNA synthetases ; Nucleic Acids Research, 50 (2022) 4029-4041.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA