Pretransfer Editing in Threonyl-tRNA Synthetase: Roles of Differential Solvent Accessibility and Intermediate Stabilization

Abstract

Aminoacyl-tRNA synthetases (aaRSs) catalyze the activation of the corresponding amino acids and attachment to their cognate tRNAs with extremely high fidelity due to pre- and post-transfer editing processes. We have computationally elucidated a pretransfer editing mechanism in yeast mitochondrial threonyl-tRNA synthetase (MST1) via the combined application of classical molecular dynamics and QM/MM-MD free energy calculations. It is concluded that the pretransfer editing mechanism against seryl-AMP occurs in two steps via an oxyanion intermediate. Importantly, its formation is central to, within the aminoacylation active site, the differential rates at which threonyl-tRNA synthetase (ThrRS) is able to hydrolyze its cognate threonyl-AMP and noncognate seryl-AMP substrates. More specifically, in contrast to that observed when threonyl-AMP is bound in the ThrRS active site, the binding of seryl-AMP enables a greater number of waters to permeate into the active site. This results in greater stabilization of the seryl-AMP derived oxyanion intermediate via hydrogen bonding such that it is thermodynamically more stable by 7–10 kcal mol–1 than is the case for threonyl-AMP. Moreover, the barrier associated with the first step of the hydrolytic pretransfer mechanism is less for seryl-AMP than for threonyl-AMP by 4–10 kcal mol–1. These results help to explain the preferential degradation of noncognate seryl-AMP over threonyl-AMP by ThrRS.