engleski

How hydrophobicity modulates amino acid discrimination by isoleucyl-tRNA synthetase?

Hydrophobic effect is considered as the main driving force for binding of small molecules to proteins. To investigate how hydrophobicity affects substrate binding and its catalysis, we used isoleucyl-tRNA synthetase (IleRS) as a model enzyme. IleRS activates (Fig 1, (1)) isoleucine (Ile) and transfers it to tRNAIle (Fig 1, (2)) 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 (Fig 1, orange/red). To tackle how substrate’s hydrophobicity modulates specificity of IleRS we analysed aminoacylation and editing of α- aminobutyrate (α-ABA) – two methylene groups smaller amino acid than Ile. We found that IleRS activated α-ABA and transferred it to tRNAIle with the similar rate constants as it does with Ile. Discrimination against α-ABA was, however, exercised at the level of Km, which is 3500-times greater than the Km for activation of Ile. Yet, even at high concentrations of α-ABA there was no accumulation of α-ABA-tRNAIle. We showed that it is a consequence of fully active IleRS pre- and post-transfer editing against α-ABA. Thus, decrease in hydrophobicity disturbs binding of amino acids to IleRS (Km effect) while the chemical steps in the synthetic and editing sites remain almost unaffected. To explore how fluorine- dependent increase in hydrophobicity affects amino acid binding and/or catalysis, we tested di- and tri- γ- fluorinated analogues of α-ABA in the reactions of IleRS (Fig 1, (1)-(5)). Interestingly, fluorination had little to no effect on the Km in activation, suggesting that, in the case of IleRS, fluorine atoms cannot compensate the loss of hydrophobic interactions caused by decreased size of α-ABA. On the other hand, fluorination reduced the rate of α-ABA activation (Fig 1, (1)) up to 5-fold and had no effect on post-transfer editing reaction of α- ABA-tRNAIle (Fig 1, (5)). Also, our data suggest that “polar hydrophobicity”, a peculiar feature of fluorine, may produce adverse effect on the interactions of fluorinated substrates with proteins. Hence, the complexity of predicting those interactions could make reengineering of aminoacyl-tRNA synthetases, for the purpose of more efficient incorporation of fluorinated amino acids into proteins, highly challenging.

Discrimination mechanisms of α-aminobutyrate and its fluorinated analogues by isoleucyl-tRNA synthetase

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