Molecular dynamics simulations of the detoxification of paraoxon catalyzed by phosphotriesterase

Abstract

Combined QM(PM3)/MM molecular dynamics simulations together with QM(DFT)/MM optimizations for key configurations have been performed to elucidate the enzymatic catalysis mechanism on the detoxification of paraoxon by phosphotriesterase (PTE). In the simulations, the PM3 parameters for the phosphorous atom were re-optimized. The equilibrated configuration of the enzyme/substrate complex showed that paraoxon can strongly bind to the more solventexposed metal ion Zn β , but the free energy profile along the binding path demonstrated that the binding is thermodynamically unfavorable. This explains why the crystal structures of PTE with substrate analogues often exhibit long distances between the phosphoral oxygen and Zn β . The subsequent S N 2 reaction plays the key role in the whole process, but controversies exists over the identity of the nucleophilic species which could be either a hydroxide ion terminally coordinated to Zn α or the μ-hydroxo bridge between the α-and β-metals. Our simulations supported the latter and showed that the rate-limiting step is the distortion of the bound paraoxon in order to approach the bridging hydroxide. After this preparation step, the bridging hydroxide ion attacks the phosphorous center and replaces the diethyl phosphate with a low barrier. Thus, a plausible way to engineer PTE with enhanced catalytic activity is to stabilize the deformed paraoxon. Conformational analyses indicate that Trp131 is the closest residue to the phosphoryl oxygen, and mutations to Arg or Gln or even Lys which can shorten the hydrogen bond distance with the phosphoryl oxygen could potentially lead to a mutant with enhanced activity for the detoxification of organophosphates.

Printed Graphene Electrochemical Biosensors Fabricated by Inkjet Maskless Lithography for Rapid and Sensitive Detection of Organophosphates

“…The biosensors inability to detect these ubiquitous phosphates is consistent with the reported class of PTE that catalyzes these pesticides at a much slower rate. 51,54,62 Additional pesticides that do not contain phenolic-leaving groups or are not catalyzed by PTE (dichlofenthion, fenitrothion, phoxim, and dimethoate) were also tested and do not show any substantial increase in oxidation current. Paraoxon was then added again to the solution, and a similar step height was observed demonstrating that the immobilized PTE was still active.…”

“…PTE degrades paraoxon by hydrolyzing the P−O bond yielding diethyl phosphate and p-nitrophenol, which has a strong absorption at 405 nm with an extinction coefficient of ∼18 000 M −1 cm −1 , opposed to paraoxon which has minimal absorption ( Figure 5a). 54 Similar to methods described, 55 three varying ratios of PTE ink (0.5, 1, and 5% which accounts for 2, 4, and 20 nM PTE) were assayed versus varying concentrations of paraoxon (1, 2, 4, and 8 μM). A standard enzymatic rate reaction model was constructed using Sigma plot's enzyme module ( Figure 5b).…”

Towards a barrier height benchmark set for biologically relevant systems

“…Thus, taking B3LYP/6-31G(d,p) as the standard, PM6 fails to predict the correct mechanism for PTE, which also explains the very large errors observed for the PM6 single point calculations in Table 1. The PTE mechanism has been studied using AM1 (Wong and Gao, 2007), PM3 (Zhang et al, 2009), and AM1/d-based QM/MM methods (López-Canut et al, 2012) and that these studies have suggested other reaction mechanisms. We emphasize that we have not explored the mechanism of PTE using PM6 but simply compared the structures resulting from the PM6 geometry optimizations initiated from the B3LYP/6-31G(d,p) geometries obtained by Chen et al (2007) In summary, geometry optimization with PM6 shows that for PTE this method predicts a different mechanism compared to B3LYP/6-31G(d,p), consistent with the fact that PM6//B3LYP/6-31G(d,p) energetics differ very significantly from the reference values as discussed above.…”

A Combination of Computational and Experimental Approaches to Investigate the Binding Behavior of B.sub Lipase A Mutants with Substrate pNPP

“…[2] There were also many similar works dedicating to uncover the mechanism of enzymatic catalysis by means of molecular modeling and quantum theory. [3][4][5] However, in most of previous studies the key residues of enzyme and the substrates are traditionally partitioned from the whole protein-ligand complexes, and then treated as a small complex entity. Although this scheme has been successfully applied in some enzyme-substrate systems, there are several disadvantages that undermine the accuracy and reliability of the computations.…”

“…For example, Yuanchao Li et al investigate the roles of anchor residues and water in the process of epitope peptides binding to HLA-A*0201 by using of QM/MM, [7] and Jans H. Alzate-Morales et al explored the binding modes and potency of the inhibitors between guanine derivatives and cyclin-dependent kinase 2(CDK2) by using QM/MM, [8] and there were also many similar works dedicating to uncover the mechanism of enzymatic catalysis by means of ONIOM. [3][4][5] In the present work, we employed ONIOM methodology and docking to explore the influence of individual amino acids of Bacillus subtilis (B.sub) lipase A on the hydrolysis reaction, with the aim to guide mutagenesis experiments on the basis of computationanl framework. After preliminary probing using PROBE program [29] a hydrophilic residue Ile12 which has intensive van der Waals contacts with the inhibitor in the complex system was selected as a hotspot.…”