The peptide bond between E292-A293 of Escherichia coli leucyl-tRNA synthetase is essential for its activity

Escherichia coli leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that contains a large connecting polypeptide (CPI) inserted int o its nucleotide binding fold, or active site. In this study, purified leuc yl-tRNA synthetase was found to be cleaved between E292 and A293 in its CP1 domain. SDS-PAGE analysis showed peptides of 63 and 34 kDa in addition to the native 97.3 kDa synthetase. By internal complementation, the two peptid es could form a 97.3 kDa complex similar to the native LeuRS. This complex could support the ATP similar to PPi exchange activity of LeuRS, but could not complement for aminoacylation. To study the function of the region arou nd the bond of E292 and A293, four pairs of peptides resulting from differe nt cleavage sites in CP1 were reconstituted in vivo. With the exception of the enzyme assembled from the E292-A293 cleavage site, all the reassembled LeuRSs catalyzed the aminoacylation of tRNA(Leu). Although the E292-A293-cl eaved LeuRS could not catalyze aminoacylation, fluorescence titration revea led that its tRNA binding ability was almost identical to that of wild-type LeuRS. These results suggest that the region around E292-A293 may be respo nsible for maintaining the proper conformation of LeuRS required for the tR NA charging activity.