Mechanism and Free Energy Landscape of tRNA Charging at the Active Site of Leucyl tRNA Synthetase: A QM/MM Simulation Study with Enhanced Sampling.

Leucyl-tRNA synthetase facilitates the transfer of leucine from leucyl-adenylate (Leu-AMP) to the 3'-terminal A76 of tRNA^Leu during the charging step of aminoacylation reaction. In the present study, we have investigated the charging step in leucyl tRNA synthetase from E. coli (Ec). Utilizing hybrid QM/MM simulations and enhanced sampling techniques, we have explored the free energy landscape of the charging reaction. An analysis of the active site organization from μs long classical molecular dynamics simulations reveals the network of interactions essential for a catalytically competent conformation. The free energy profile obtained from QM/MM simulations unveils a two-step process where the amino group (NH₃⁺) of Leu-AMP serves as a general base. The first step of the charging process involves a proton transfer from O2' of A76 to the amino group of the leucyl-adenylate following the deprotonation of the positively charged amino group (NH₃⁺) to the carboxylic oxygen of Asp80, with a free energy barrier of 4.2 kcal mol^-1. In the second step, the deprotonated O2' attacks the carbonyl carbon (C) and breaks the C-O3α bond of the Leu-AMP. This step exhibits a free energy barrier of 22.9 kcal mol^-1 with a tetrahedral structure in the transition state. This study uncovers the charging pathway for leucyl tRNA synthetase in E. coli, with no water involvement in the charging step. It may be noted that the charging step varies between class I LeuRS (binding to 2'-OH of A76) and class II AspRS (binding to 3'-OH of A76). Despite these distinctions, both classes share a common pathway, highlighting the amino group's role as a base and the aspartate residue (Asp80 of LeuRS^Ec and Asp233 of AspRS^Ec) as the proton acceptor.