An aminoacyl-tRNA synthetase with a defunct editing site.
Many aminoacyl-tRNA synthetases (aaRSs) contain two active sites, a synthetic site catalyzing aminoacyl-adenylate formation and tRNA aminoacylation and a second editing or proofreading site that hydrolyzes misactivated adenylates or mischarged tRNAs. The combined activities of these two sites lead to rigorous accuracy in tRNA aminoacylation, and both activities are essential to LeuRS and other aaRSs. Here, we describe studies of the human mitochondrial (hs mt) LeuRS indicating that the two active sites of this enzyme have undergone functional changes that impact how accurate aminoacylation is achieved. The sequence of the hs mt LeuRS closely resembles a bacterial LeuRS overall but displays significant variability in regions of the editing site. Studies comparing Escherichia coli and hs mt LeuRS reveal that the proofreading activity of the mt enzyme is disrupted by these sequence changes, as significant levels of Ile-tRNA(Leu) are formed in the presence of high concentrations of the noncognate amino acid. Experiments monitoring deacylation of Ile-tRNA(Leu) and misactivated adenylate turnover revealed that the editing active site is not operational. However, hs mt LeuRS has weaker binding affinities for both cognate and noncognate amino acids relative to the E. coli enzyme and an elevated discrimination ratio. Therefore, the enzyme achieves fidelity using a more specific synthetic active site that is not prone to errors under physiological conditions. This enhanced specificity must compensate for the presence of a defunct editing site and ensures translational accuracy.
Lue
Stanley W
SW
Eugene F. Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Kelley
Shana O
SO
D000215:Acylation; D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017393:RNA Editing; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012356:RNA, Transfer, Leu / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D012329:RNA, Bacterial; D012356:RNA, Transfer, Leu; D011994:Recombinant Proteins; D000604:Amino Acyl-tRNA Synthetases