Alternative substrate-assisted hydrolysis pathways of posttransfer editing by prokaryotic leucyl-tRNA synthetase

Leucyl-tRNA synthetase (LeuRS) activates cognate leucine and wrongly activates norvaline (and other proteinogenic and non-proteinogenic amino acids), but then mischarges tRNA. Here, we studied the editing mechanisms of Thermus thermophilus LeuRS (TthLeuRS) by combination of biochemical and computational investigations with the appropriate substrate, norvalyl-tRNA, as previously reported for E. coli LeuRS (EcLeuRS). Based on DFT (density functional theory) calculations, we have proposed three alternative hydrolysis mechanisms. These mechanisms differ according to which group of water molecules present in the editing site of the enzyme-substrate complex participates in the chemical reaction of ester bond cleavage. The main feature of the proposed deacylation pathways is direct assistance by the substrate in the hydrolysis process. In all three cases, the 3'-OH group of the substrate directly participates in the reaction. To confirm the proposed models, the experimental substitution of the 76 3'-OH group of the tRNA^Leu was constructed yielding a tRNA that is devoid of editing activity. QM and metadynamics have shown that hydrolysis occurs via a common underlying catalysis mechanism involving more than one water molecule. An important element of hydrolysis is the involvement of several amino acid residues of the active centre of enzymatic editing (Asp-347/Asp-344 and Thr-247) in the process of catalytic deacylation. In conclusion, it was noted that the possibility of several alternative hydrolysis pathways may indicate that a sufficiently protected and flexible error-editing mechanism has been implemented for prokaryotic leucyl-tRNA synthetase.