Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that remarkable facilitate the aminoacylation process during translation. With a high fidelity, the mischarged tRNA is prevented through implementing pre- and post-transfer proofreading mechanisms. For instance, Lysine-tRNA synthetase charges the native substrate, lysine, to its cognate tRNA. In spite of the great structural similarity between lysine to the noncognate and toxic ornithine, with the side chain of lysine being only one methylene group longer, LysRS is able to achieve this discrimination with a high efficiency. In this work, the hybrid quantum mechanics/molecular mechanics (QM/MM) investigation was applied to probe the pre-transfer editing mechanism catalyzed by lysyl-tRNA synthetase to reject the noncognte aminoacyl, L-ornityl (Orn), compared to the cognate substrate, L-lysyl. Particularly, the self-cyclization pre-transfer editing mechanism was explored for the two substrates. The substrate-assisted self-cyclization editing of Orn-AMP, where its phosphate moiety acts as the catalytic base, is found to be the rate-determining step with an energy barrier of 101.2 kJ mol-1. Meanwhile, the corresponding rate-limiting pathway for the native Lys-AMP lies at 140.2 kJ mol-1. This observation clearly indicated the infeasibility of this catalytic scenario in the presence of the native substrate. Interestingly, a thermodynamically favorable cyclic product of -92.9 kJ mol-1 with respect to the aminoacyl reactant complex demonstrated evidence of a successful pre-transfer editing. This reaction resulted in the discharge of the on-cognate -ornithine derivative from LysU's active site. These valuable mechanistic insights are valuable to enrich our knowledge of this extremely efficient and specific catalytic machinery of LysRS.Communicated by Ramaswamy H. Sarma.
Keywords: Aminoacyl-tRNA synthetase; LYSRS; LYSU; QM/MM; catalytic mechanism; molecular dynamics; multi-scale.