engleski

What tailors discrimination against smaller hydrophobic amino acids in isoleucyl-tRNA synthetase?

Hydrophobic effect is considered as the main driving force for binding of small molecules to proteins. To investigate how size and hydrophobicity affect substrate binding and its catalysis, we used isoleucyl-tRNA synthetase (IleRS) as a model enzyme. IleRS activates isoleucine (Ile) and transfers it to tRNAIle for participation in protein synthesis. IleRS can also misactivate non-cognate valine (Val) and norvaline (Nva), both being one methylene group smaller than Ile, and transfer them to tRNAIle. To prevent accumulation of misacylated tRNAs, and thus error in protein synthesis, IleRS employs pre- and post- transfer hydrolytic editing. To tackle how substrate’s size and hydrophobicity modulate specificity of IleRS we analyzed aminoacylation and editing of α-aminobutyrate (α-ABA) – two methylene groups smaller amino acid than Ile. We found that IleRS activated α-ABA 7100-times less efficiently than Ile with most of the effect (3500-times) coming from the increase in the Km value. No discrimination against α-ABA was observed at the transfer step. Yet, α-ABA- tRNAIle has not been accumulated by the enzyme even at high concentrations of used α-ABA. Using detailed kinetic analysis, we showed that it was a consequence of active IleRS’s pre- and post- transfer editing against α-ABA. Thus, decrease in size and hydrophobicity disturbed binding of amino acids to IleRS (Km effect) while the chemical steps in the synthetic and editing sites remained almost unaffected. To examine whether α-ABA binding was impaired by loss of Van der Waals interactions due to fewer methylene groups or by decrease in entropic contribution to binding, we tested more hydrophobic, di- and tri-γ- fluorinated, analogues of α-ABA in the reactions of IleRS. Interestingly, fluorination had little to no effect on the Km in activation, suggesting that, in the case of IleRS, hydrophobicity is not the main driving force for substrate binding. Furthermore, fluorination reduced the rate of α- ABA activation up to 5-fold and had no effect on post-transfer editing reaction of α-ABA-tRNAIle. This suggest that “polar hydrophobicity”, a peculiar feature of fluorine, may produce opposing effects on the binding and catalysis of fluorinated substrates. Hence, the reengineering of aminoacyl-tRNA synthetases, for the purpose of more efficient incorporation of fluorinated amino acids into proteins, appears to be highly challenging.

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

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