2008 Eighth Annual Beckman Scholars Symposium
Thursday Poster Session - July 27, 2006

Aminoacyl-tRNA synthetases activate specific amino acids and transfer them onto cognate tRNA molecules. Due to the similarity in amino acid side chains, synthetases sometimes misactivate non-cognate amino acids and misacylate their cognate tRNAs. Several pathways for correcting errors in amino acid activation (pre-transfer editing) and aminoacylation (post-transfer editing) have been proposed. Although it is well established that post-transfer editing occurs via hydrolysis of the aminoacyl ester bond in an active site that is distinct from the site of adenylate synthesis, the location and mechanism of pre-transfer editing are less well understood. Class II prolyl-tRNA synthetases (ProRS) has previously been shown to weakly misactivate non-cognate alanine and to carry out tRNA-independent pre-transfer editing. Therefore, the mechanism of pre-transfer editing by Escherichia coli ProRS was investigated. Comparison of the rates of ATP hydrolysis by ProRS stimulated in the presence of non-cognate alanine and cognate proline, as well as the non-catalytic solution hydrolysis of Ala-AMP and Pro-AMP, suggests that the editing reaction is not enzyme catalyzed. The relative binding affinities estimated for cognate and non-cognate non-hydrolyzable adenylate analogs show that ProRS binds the alanine analog ~20-fold less tightly than the corresponding proline analog. Kinetic data also show that there is a direct correlation between the KM measured for various proline analogs and their relative rates of pre-transfer editing. Taken together, these data support a non-enzymatic, or selective hydrolysis, model for pre-transfer editing wherein the non-cognate substrate is more readily released from the enzyme’s active site and hydrolyzed in solution.