Naslov
Discrimination of α-aminobutyrate and its fluorinated analogues in the synthetic and editing reactions of isoleucyl-tRNA synthetase

Autori
Živković, Igor ; Gruić Sovulj, Ita

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

Izvornik
Molecular biophysics : ABC of the puzzle of life, book of abstracts / Ivošević DeNardis, Nadica ; Campos-Olivas, Ramon ; Miele, Adriana E. ; England, Patrick ; Vuletić, Tomislav - Zagreb : Institut Ruđer Bošković ; Croatian Biophysical Society, 2019, 142-143

ISBN
978-953-7941-28-4

Skup
3rd COST-sponsored ARBRE-MOBIEU plenary meeting Molecular Biophysics - ABC of the puzzle of Life

Mjesto i datum
Zagreb, Hrvatska, 18.03.2019. - 20.03.2019

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
Isoleucyl-tRNA synthetase, alpha-aminobutyrate, fluorinated amino-acids, hydrophobicity

Sažetak
Isoleucyl-tRNA synthetase (IleRS) is an enzyme that catalyzes activation of isoleucine (Ile) (Fig, (1)) and its transfer to tRNAIle (Fig, (2)). Synthetized Ile-tRNAIle is a substrate for protein synthesis. It has been shown that IleRS can also misactivate and transfer to tRNAIle non-cognate amino acids valine (Val) and norvaline (Nva), both being one methylene group smaller than Ile. Nva is a non- proteinogenic amino acid that sporadically accumulates in cells. To prevent accumulation of misacylated tRNAs, and thus the error in protein synthesis, IleRS employs pre- and post- transfer hydrolytic editing (Fig, orange/red). In order to explore how substrate hydrophobicity affects amino acid discrimination in the synthetic and editing reactions of IleRS, we now tested non-cognate α- aminobutyrate (α-ABA), which is two methylene groups smaller than Ile. α-ABA is also a metabolic amino acid that accumulates in cells. IleRS activated and transferred α-ABA to tRNAIle with the similar rate constants as obtained with Ile. However, IleRS exercised discrimination against α- ABA predominantly at the level of Km, which is 3500-times greater than the Km for Ile. This suggests that binding of α-ABA to the active site of IleRS is impaired. Despite α-ABA being activated and transferred to tRNAIle, there was no accumulation of α-ABA-tRNAIle due to active IleRS pre- and post- transfer editing against α-ABA. Kinetic analysis showed that post- transfer editing is rapid and dominant editing reaction, as previously found for Val and Nva. Our data revealed that decrease in hydrophobicity mainly affects the amino acid binding to IleRS (Km effect) while the effects on the rate constants of the synthetic and editing reactions were modest. To explore whether the increase in hydrophobicity by introducing fluorine will affect α-ABA discrimination by IleRS, we investigated aminoacylation and editing of di- and tri- γ- fluorinated analogues of α-ABA. Introduction of fluorine reduced the activation rate up to 5-fold, while surprisingly it had little to no effect on the amino acid binding (Km) and editing reactions. Thus, in the case of IleRS, fluorination of α-ABA does not compensate the loss of hydrophobic interactions caused by decrease in the size of the amino acid substrate. Even more, our data suggest that “polar hydrophobicity”, a peculiar feature of fluorine, produced adverse effect on the binding of fluorinated α-ABA to IleRS. Hence, the unpredictability of fluorine-enzyme interactions could make reengineering of aminoacyl-tRNA synthetases, for the purpose of more efficient incorporation of fluorinated amino acids into proteins, more challenging than anticipated.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

POVEZANOST RADA