RESUMO
Organophosphorus compounds (OP) are part of a group of compounds that may be hazardous to health. They are called neurotoxic agents because of their action on the nervous system, inhibiting the acetylcholinesterase (AChE) enzyme and resulting in a cholinergic crisis. Their high toxicity and rapid action lead to irreversible damage to the nervous system, drawing attention to developing new treatment methods. The diisopropyl fluorophosphatase (DFPase) enzyme has been considered as a potent biocatalyst for the hydrolysis of toxic OP and has potential for bioremediation of this kind of intoxication. In order to investigate the degradation process of the nerve agents Tabun, Cyclosarin and Soman through the wild-type DFPase, and taking into account their stereochemistry, theoretical studies were carried out. The intermolecular interaction energy and other parameters obtained from the molecular docking calculations were used to construct a data matrix, which were posteriorly treated by statistical analyzes of chemometrics, using the PCA (Principal Components Analysis) multivariate analysis. The analyzed parameters seem to be quite important for the reaction mechanisms simulation (QM/MM). Our findings showed that the wild-type DFPase enzyme is stereoselective in hydrolysis, showing promising results for the catalytic degradation of the neurotoxic agents under study, with the degradation mechanism performed through two proposed pathways.
Assuntos
Substâncias para a Guerra Química/metabolismo , Hidrolases de Triester Fosfórico/metabolismo , Biodegradação Ambiental , Organofosfatos/metabolismo , Compostos Organofosforados/metabolismo , Análise de Componente Principal , Soman/metabolismoRESUMO
The bacterial enzyme organophosphorus hydrolase (OPH) exhibits both catalytic and substrate promiscuity. It hydrolyzes bonds in a variety of phosphotriester (P-O), phosphonothioate (P-S), phosphofluoridate (P-F), and phosphonocyanate (F-CN) compounds. However, its catalytic efficiency varies markedly for different substrates, limiting the broad-range application of OPH as catalyst in the bioremediation of pesticides and chemical war agents. In the present study, pK(a) calculations and multiple explicit-solvent molecular dynamics (MD) simulations were performed to characterize and contrast the structural dynamics of OPH bound to two substrates hydrolyzed with very distinct catalytic efficiencies: the nerve agent soman (O-pinacolylmethylphosphonofluoridate) and the pesticide paraoxon (diethyl p-nitrophenyl phosphate). pK(a) calculations for the substrate-bound and unbound enzyme showed a significant pK(a) shift from standard values (ΔpK(a) = ±3 units) for residues His254 and Arg275. MD simulations of protonated His254 revealed a dynamic hydrogen bond network connecting the catalytic residue Asp301 via His254 to Asp232, Asp233, Arg275, and Asp235, and is consistent with a previously postulated proton relay mechanism to ferry protons away from the active site with substrates that do not require activation of the leaving group. Hydrogen bonds between Asp301 and His254 were persistent in the OPH-paraoxon complex but not in the OPH-soman one, suggesting a potential role for such interaction in the more efficient hydrolysis of paraoxon over soman by OPH. These results are in line with previous mutational studies of residue His254, which led to an increase of the catalytic efficiency of OPH over soman yet decreased its efficiency for paraoxon. In addition, comparative analysis of the molecular trajectories for OPH bound to soman and paraoxon suggests that binding of the latter facilitates the conformational transition of OPH from the open to the closed substate promoting a tighter binding of paraoxon.
Assuntos
Arildialquilfosfatase/metabolismo , Paraoxon/metabolismo , Soman/metabolismo , Arildialquilfosfatase/química , Arildialquilfosfatase/genética , Bactérias/enzimologia , Ligação de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Mutação , Paraoxon/química , Ligação Proteica , Soman/químicaRESUMO
Many organophosphorus compounds (OPs) are potent cholinesterase inhibitors, accounting for their use as insecticides and, unfortunately, also as nerve agents. Each year there are approximately 3 million pesticide poisonings world-wide resulting in 220,00 deaths. In 1990, there were 1.36 million kg of chlorpyrifos, 4.67 million kg of diazinon and 1.23 million kg of ethyl parathion manufactured in the USA (data supplied by the USEPA). In addition to exposure risks during pesticide manufacturing, distribution and use, there are risks associated with the major international effort aimed at destroying the arsenals of nerve agents, including soman and sarin. The United States has pledged to destroy approximately 25,000 tons of chemical agents by the end of the decade. The high density lipoprotein (HDL)-associated enzyme paraoxonase (PON1) contributes significantly to the detoxication of several OPs (Fig. 1). The insecticides parathion, chlorpyrifos and diazinon are bioactivated to potent cholinesterase inhibitors by cytochrome P-450 systems. The resulting toxic oxon forms can be hydrolysed by PON1, which also hydrolyses the nerve agents soman and sarin (Fig. 1). PON1 is polymorphic in human populations and different individuals also express widely different levels of this enzyme. The Arg192 (R192) PON1 isoform hydrolyses paraoxon rapidly, while the Gln192 (Q191) isoform hydrolyses paraoxon slowly. Both isoforms hydrolyse chlorpyrifos-oxon and phenylacetate at approximately the same rate. The role of PON1 in OP detoxication is physiologically significant. Injected PON1 protects against OP poisoning in rodent model systems and interspecies differences in PON1 activity correlate well with observed median lethal dose (LD50) values. We report here a simple enzyme analysis that provides a clear resolution of PON1 genotypes and phenotypes allowing for a reasonable assessment of an individual's probable susceptibility or resistance to a given OP, extending earlier studies on this system. We also show that the effect of the PON1 polymorphism is reversed for the hydrolysis of diazoxon, soman and especially sarin, thus changing the view of which PON1 isoform is considered to be protective.