Enzymatic P–O Bond Cleavage: Criteria of Dissociative and Associative Mechanisms

Nucleophilic substitution at phosphorus centers is among the most widely spread biochemical reactions of natural and artificial organophosphates. These reactions can occur via dissociative and associative mechanisms, depending on a particular enzyme and substrate. The most straightforward yet expensive way to determine the mechanism is to calculate the energy profile for the entire reaction. In this study, we analyzed a set of 15 enzymes that cleave P-O bonds in different organophosphates using QM/MM methods on potential energy surfaces and in MD simulations, combined with subsequent electron density analysis. We demonstrate that the structure and electron density features of the reaction region in the enzyme-substrate complex determine the mechanism of the subsequent reaction. The cleaving P-O_LG bond is elongated in systems that undergo chemical reactions via a dissociative mechanism compared with the associative one. Importantly, the bond length distribution remains the same upon changing the DFT functional in simulations. Likely, the P-O_LG bond length is mostly determined by the protein environment rather than the nature of the nucleophile: a virtual change of the neutral water molecule to the hydroxide anion does not shift the distance distribution. The cleaving P-O_LG bond order, calculated from electron density, is also larger for systems operating via an associative pathway. Both distances and bond orders change continuously, and it is not evident what the border value that discriminates different mechanisms is. Therefore, we propose a binary classifier that relies on the Laplacian of the electron density in the cleaving P-O_LG bond region. Electron density concentration is observed only for systems that perform reactions via an associative pathway. This criterion was tested on three adenylate kinases. Despite their structural differences, we obtained the same dissociative reaction mechanism type, which further proves the reliability of the suggested criterion.