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In the present L-valine·IleRS complex structure, a second L-valine molecule was identified at the bottom of the deep cleft in the Ins-2 domain (Fig. 3, A and B). In contrast, in the L-isoleucine·IleRS complex structure, no electron density was observed in this second pocket. Therefore, the second pocket on the Ins-2 domain is specific for L-valine, indicating that this pocket is the site for the second, fine sieve of the double-sieve mechanism. The space made up of the invariant Trp232 and Tyr386 residues is just large enough to accommodate L-valine and therefore is too small for L-isoleucine (Fig. 3B). To demonstrate that the cleft on the Ins-2 domain actually functions as the hydrolytic editing site, they constructed an IleRS mutant [(219 to 265)] that lacks 47 amino acid residues, including the Trp232 of the L-valine-specific pocket. This deletion mutation completely abolished the Val- tRNAIle editing activity (Table 2) and confirmed that the cleft on the Ins-2 domain functions as the catalytic site for specific editing against Val.
Once bound, how are the valylated products hydrolyzed? Near the bound L-valine, Thr228, Thr230, Thr233, and Asn237 from the first segment and His319 from the second segment are located in close proximity (Fig. 4), which is reminiscent of the catalytic triads of hydrolases. Further mutagenesis analyses were made on the E. coli IleRS. Ala mutations of Thr243 and Asn250, which correspond to Thr230 and Asn237, respectively, of T. thermophilus IleRS, completely abolished editing activity against Val, with little change in aminoacylation activity. These mutagenesis results confirm the identity of this cleft as the editing site and the catalytic importance of both Thr230 and Asn237.