Base Mechanism to the Hydrolysis of Phosphate Triester Promoted by the Cd2+/Cd2+ Active site of Phosphotriesterase: A Computational Study.

In the present work, density functional theory (DFT) calculations at the B3LYP/6-31+G(d) and including dispersion effects were used to investigate the hydrolysis of paraoxon, using a cluster model of the active site of Cd2+/Cd2+-phosphotriesterase (PTE) from Pseudomonas diminuta. The mechanism proposed here consist of (i) Exchange of the coordinated water molecule and coordination of the substrate to the more solvent exposed Cdβ center in monodentate fashion, (ii) protonation of the μ-hydroxo bridge by the uncoordinated water molecule and in situ formation of the nucleophile, (iii) formation of a pentacoordinate intermediate with significant bond breaking to the leaving group and bond formation to the nucleophile, and (iv) protonation of the Asp301 residue and restoration of the active site through the coordination of another water molecule of the medium. The water molecules initially coordinated to the active site play a crucial role in stabilizing the transition states and the pentacoordinate intermediate. The reaction takes place in a two-step (AN + DN) mechanism, with energy barriers of 12.9 and 1.9 kcal/mol for the first and second steps, respectively, computed at the B3LYP-D3/6-311++G(2d,2p) level of theory, in excellent agreement with the experimental findings. Dispersion effects alone contribute to diminish the energy barriers as much as 26%. The base mechanism for the Cd2+/Cd2+-PTE proposed here, in conjunction with the agreement found with the experimental energetic value for the energy barrier, makes it a consistent and kinetically viable mechanistic proposal for the hydrolysis of phosphate triesters promoted by the Cd2+ substituted PTE enzyme.