Martí Sergio, Bastida Agatha, Świderek Katarzyna
Departament de Química Física i Analítica, Universitat Jaume I, Castelló de La Plana, Spain.
Departamento de Química Bio-orgánica, Instituto de Química Orgánica General (CSIC), Madrid, Spain.
Front Chem. 2019 Jan 29;6:660. doi: 10.3389/fchem.2018.00660. eCollection 2018.
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.
这项工作聚焦于对腺苷酸基团（5'-单磷酸腺苷）从三磷酸腺苷（ATP）转移至抗生素的OH-4'羟基的机理研究。运用量子力学/分子力学（QM/MM）混合技术，我们研究了由4'-O-核苷酸转移酶[ANT(4')]催化的卡那霉素A（KAN）失活过程的底物和碱基辅助机制，该酶对几乎所有氨基糖苷类抗生素都有活性。在M06-2X/MM理论水平下，通过自由能微扰方法获得的自由能面表明，最有利的反应路径呈现出12.2 kcal·mol的能垒，这对应于Glu145对KAN的O4'的协同活化。此外，还计算了ATP的桥连O3α以及非桥连O1α、O2α和O5'原子的一级和二级氧动力学同位素效应（KIEs）。对于Glu145辅助机制，观察到的正常1°-KIE为1.2%，2°-KIE为0.07%，与实验测量数据非常吻合。此外，基于所得结果，讨论了静电和压缩效应在酶催化中的作用。
