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. 2014 May 1;118(17):4479-89.
doi: 10.1021/jp410422c. Epub 2014 Apr 21.

Hydrolysis of DFP and the nerve agent (S)-sarin by DFPase proceeds along two different reaction pathways: implications for engineering bioscavengers

Troy Wymore  1 , Martin J Field, Paul Langan, Jeremy C Smith, Jerry M Parks
Affiliations

Hydrolysis of DFP and the nerve agent (S)-sarin by DFPase proceeds along two different reaction pathways: implications for engineering bioscavengers

Troy Wymore et al. J Phys Chem B. .

Abstract

Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents ("bioscavengers") is an emerging prophylactic approach to diminish their toxic effects. Although its native function is not known, diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P-F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca(2+), donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily because of the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed β-propeller folds for more efficient degradation of OP compounds.

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Figures

Figure 1
Figure 1
Structural diagrams of selected nerve agents. An asterisk denotes a chiral center.
Figure 2
Figure 2
(Left) Proposed mechanism for phosphoenzyme intermediate formation involving Asp229 as the nucleophile. (i) Asp229 attacks the phosphorus center of DFP to form a pentavalent intermediate and (ii) the P–F bond dissociates to form a tetrahedral phosphoenzyme intermediate. Hydrolysis of the phosphoenzyme intermediate is not shown. (Right) Proposed mechanism for hydrolysis involving an activated water as the nucleophile. (i) Asp229 abstracts a proton from a water molecule either stepwise or in concert as (ii) water attacks the phosphorus center, (iii) Glu21 abstracts a proton either stepwise or in concert as (4) water forms a bond with phosphorus, and (iv) the P–F bond dissociates.
Figure 3
Figure 3
Structural depiction of DFPase showing each blade of the six-bladed β-propeller fold with the active site residue from each blade shown in the CPK representation. Black spheres represent the catalytic (foreground) and structural (background) Ca2+ ions.
Figure 4
Figure 4
(A) DFT/MM US free energy profile for the An + Dn reaction of DFPase with DFP substrate. The reaction coordinate is defined as the mass-weighted distance difference between Oδ(Asp229)–P and P–F and progresses from the Michaelis complex though the first transition state (TS-1) to the pentavalent phosphoenzyme intermediate and then through the second transition state (TS-2) to the tetrahedral phosphoenzyme intermediate at a reaction coordinate value beyond 1.4. Statistical errors range from 0.01 kcal mol–1 near the Michaelis complex to 0.05 kcal mol–1 near the tetrahedral phosphoenzyme intermediate. (B) Representative snapshot of the Michaelis complex. (C) Representative snapshot of the pentavalent phosphoenzyme intermediate. (D) Representative snapshot of the rate-limiting transition state in which fluoride dissociates from the pentavalent intermediate. Note that Ca2+-coordinating residues Asn120 and Asn175 are omitted in panels B–D for clarity.
Figure 5
Figure 5
(A) DFT/MM US free energy profile for the nucleophilic attack of an activated water on the phosphoenzyme intermediate. The statistical error ranged from 0.01 to 0.04 kcal mol–1. (B) Snapshot showing the interactions that stabilize hydroxide, which is separated from Cδ of Asp229 by 3.1 Å. (C) Enzyme-bound phosphoenzyme hydrolysis product (diisopropyl phosphate). Note that Ca2+-coordinating residue Asn120 is omitted in panels B and C for clarity.
Figure 6
Figure 6
(Left) DFT/MM US free energy profile for the AnDn reaction of DFPase with (S)-sarin. The arrow from the DFP:DFPase pentavalent intermediate basin to the corresponding point in the DFPase:(S)-sarin free energy profile is shown to highlight the complete lack of this intermediate in the reaction with (S)-sarin. Statistical errors range from 0.01 kcal mol–1 near the Michaelis complex to 0.06 kcal mol–1 near the tetrahedral phosphoenzyme intermediate. (Right) Representative snapshot of the transition state from the DFT/MM US simulation of the DFPase:(S)-sarin reaction.
Figure 7
Figure 7
Snapshot from DFT/MM simulations of DFPase/(S)-sarin supporting a nucleophilic attack on the phosphorus center of (S)-sarin hydrolysis by a water molecule upon activation by proton transfer to Asp229. Selected distances shown are shown in angstroms. Ca2+-coordinating residues Asn120 and Asn175 are omitted for clarity.
Figure 8
Figure 8
Gas-phase potential energy scans obtained by varying the acetate–O–P distance in (left) acetate–DFP and (right) acetate–sarin obtained with the BP86, B3LYP, mPWPW91, and mPW1PW91 functionals and the 6-31+G(d) basis set.
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