engleski

Opposing activities share the same active site: synthesis and breakdown of aminoacyl-adenylates by isoleucyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that covalently attach amino acids to cognate tRNAs in a two step reaction occurring within the synthetic site. Amino acid is firstly activated via formation of aminoacyl-adenylate. In the second step, aminoacyl-moiety is transferred to cognate tRNA. Since cells contain many different tRNAs and amino acids, aaRSs have a difficult task of selecting the correct substrates. While selection of cognate tRNA does not pose a serious problem due to large interacting surface, discrimination of amino acids, especially structurally similar ones, is more difficult to achieve. Isoleucyl-tRNA synthetase (IleRS) is an aaRS which, besides cognate isoleucine, efficiently activates noncognate valine. Moreover, it was shown that transfer of valine to tRNAIle proceeds with the same rate as the transfer of isoleucine. To ensure accuracy of protein biosynthesis, IleRS hydrolyzes valyl-adenylate by pre-transfer editing, and misacylated tRNA by post-transfer editing. While it is well known that post-transfer editing occurs in a spatially separate editing domain, location and mechanisms of pre-transfer editing have been a matter of debate. Although pre-transfer editing by IleRS occurs in the absence of tRNA, it is significantly stimulated by its presence. We demonstrated that both the tRNA-independent and the tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme, which strongly suggests that pre-transfer editing reaction is occurring in the synthetic site. Further, we showed that the balance between pre-transfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate between hydrolysis and transfer. To unveil the mechanism of the pre-transfer editing, we mutated several conserved residues within the synthetic site. Obtained mutants also enabled us to gain deeper insight into cognate and noncognate synthetic reactions. Mutations of residues responsible for binding ribose-moiety of ATP resulted in at least 100-fold decrease in kcat for amino acid activation. These mutants had barely detectable hydrolytic activity, possibly, but not necessarily, due to the observed slow activation. On the other hand, mutations of residues contacting isoleucine primarily increased Km in activation. These mutants generally displayed increased hydrolytic activity towards both isoleucyl- and valyl-adenylate. Our data suggest that synthesis and breakdown of aminoacyl-adenylates are intertwined within the synthetic site, presumably as a consequence of highly overlapping sites of occurrence. Therefore, it seems that the synthetic site of isoleucyl-tRNA synthetase evolved as a compromise between efficiency of cognate amino acid activation and precision of noncognate aminoacyl-adenylate hydrolysis.

aminoacyl-tRNA synthetase; proofreading; pre-transfer editing

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