What roles do the residue Asp229 and the coordination variation of calcium play of the reaction mechanism of the diisopropyl-fluorophosphatase? A DFT investigation

Abstract

Phosphotriesterase enzyme diisopropyl-fluorophosphatase (DFPase) from Loligo vulgaris utilizes one Ca2+ ion to efficiently catalyze the hydrolysis of the substrate diisopropyl fluorophosphate and a wide range of organophosphorus nerve agents, including soman, sarin, and tabun. In the present work, the catalytic mechanism of DFPase is investigated using the hybrid density functional theory method B3LYP with a large quantum chemical model of the active site abstracted from the X-ray crystal structure. For the first step, two different pathways were considered: (1) residue Asp229 as a nucleophile in-line attacks on the phosphorus center and (2) an activated water molecule as the nucleophile attacks on the phosphorus center. Both the Asp229 and the activated water molecule are capable of proceeding nucleophilic attack on the substrate in the presence of Ca2+ ion with the associated barriers 14.8 and 6.0 kcal/mol, respectively. The latter is much easier to perform the nucleophile attack. From the phosphoenzyme intermediate with the hexa-coordinated Ca2+, the uncoordinated Glu21 functions as a general base activated an additional water molecule to attack the carbon center of Asp229 and make the phosphate release. Residues Asn120 and Asn175 promote the elimination of the fluoride via donating strong hydrogen bonds. Residue Asp229 plays a dual role during the hydrolysis reaction process, either as a nucleophile or as a general base to activate the water nucleophile. The role of the calcium ion is providing a necessary conformation of the active site, facilitating the nucleophile formation and substrate orientation. Our calculated results shed further light on the organophosphate hydrolysis mechanism and guiding for structure-based protein engineering to modify hydrolysis rates and substrate specific.