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

much easier to perform the nucleophile attack. From the phosphoenzyme intermediate with the hexa-coordinated Ca 2+ , 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.

Similar Active Sites and Mechanisms Do Not Lead to Cross-Promiscuity in Organophosphate Hydrolysis: Implications for Biotherapeutic Engineering

“… 47 , 48 However, these studies do not conclusively discriminate between both mechanisms because they either did not compare the general base and nucleophilic mechanisms for the same substrate 47 or obtained reaction energies so low as to be physically unrealistic. 48 In addition, neither study explored the calculated energetics of the corresponding uncatalyzed reaction in aqueous solution. However, examining the corresponding uncatalyzed reaction at the same level of theory as the enzyme-catalyzed reaction is absolutely critical in order to quantify the catalytic effect of the enzyme through different pathways.…”

“…DFPase also has a much more accessible active site than PON1, 19 lacking the three helices that decorate the top of the β-propeller in PON1 and help it interact with HDL, thus also sequestering the active site from solvent. 19 , 32 , 34 In addition, although DFPase is far less studied than PON1, there has nevertheless also been interest in developing engineered variants of this enzyme that can be used as a biotherapeutic, 45 and the enzyme has been the subject of several biochemical, structural and also more recently computational studies (e.g., refs ( 1 ), ( 36 ), ( 39 ), ( 44 ), and ( 46 − 48 )).…”

Theoretical Studies Applied to the Evaluation of the DFPase Bioremediation Potential against Chemical Warfare Agents Intoxication

“…In this context, the employment of enzymes capable of degrading OP shows up as an alternative treatment [ 11 ]. Among the enzymes investigated with this applicability, the Diisopropyl fluorophosphatase (DFPase), from the squid Loligo vulgaris , has emerged recently, showing a good potential for this purpose [ 12 ].…”

“…Previous studies have suggest an essential role of the Asp229 residue in the DFPase catalytic activity, since it has the correct orientation to perform a nucleophilic attack in the hydrolysis mechanism through bimolecular nucleophilic substitution (S N 2), and it is also coordinated to the Ca 2+ ion together with other residues and water molecules [ 7 , 11 , 12 , 13 ]. One possible pathway for the catalysis is that the Asp229 acts as a nucleophile, attacking the OP coordinated to the Ca 2+ ion.…”

“…The Glu21 participates in these proton transfers. The leaving group is released and the hydrolyzed substrate is removed from the Ca 2+ ion at the ionized form as the reaction product [ 7 , 11 , 12 , 13 ]. Simplified versions of both these proposed mechanisms are represented in Figure 3 .…”