Theoretical Studies on Mechanism of Inactivation of Kanamycin A by 4'-O-Nucleotidyltransferase.

This work is focused on mechanistic studies of the transfer of an adenylyl group (Adenoside-5'-monophosfate) from adenosine 5'-triphosphate (ATP) to a OH-4' hydroxyl group of an antibiotic. Using hybrid quantum mechanics/molecular mechanics (QM/MM) techniques, we study the substrate and base-assisted mechanisms of the inactivation process of kanamycin A (KAN) catalyzed by 4'-O-Nucleotidyltransferase [ANT(4')], an active enzyme against almost all aminoglycoside antibiotics. Free energy surfaces, obtained with Free Energy Perturbation methods at the M06-2X/MM level of theory, show that the most favorable reaction path presents a barrier of 12.2 kcal·mol that corresponds to the concerted activation of O4' from KAN by Glu145. In addition, the primary and secondary O kinetic isotope effects (KIEs) have been computed for bridge O3α, and non-bridge O1α, O2α, and O5' atoms of ATP. The observed normal 1°-KIE of 1.2% and 2°-KIE of 0.07% for the Glu145-assisted mechanism are in very good agreement with experimentally measured data. Additionally, based on the obtained results, the role of electrostatic and compression effects in enzymatic catalysis is discussed.