Inactivation of cytochrome P450 3A during the oxidative desulfuration of methyl parathion.

摘要

The organophosphorus insecticides are responsible for more human poisonings than are any other class of insecticides. The organophosphate pesticides are known to inactivate members of the cytochrome P450 family of enzymes and are categorized as mechanism-based inhibitors. Mechanism-based inhibitors are substrates that give rise to reactive intermediates that interact with the enzyme and cause a loss of function. Methyl parathion and other organophosphate pesticides feature a thio-phosphate moiety and are subject to oxidative desulfuration by cytochrome P450. The sulfur liberated during the oxidative desulfuration of organo-sulfur compounds is reported to bind to cysteine residues of the P450 enzyme.; The experiments performed in this study exhibited kinetics of metabolite formation consistent with the loss of enzyme activity resulting from the metabolism of methyl parathion. Patterns of testosterone hydroxylation subsequent to incubation with methyl parathion were consistent with mechanism-based inhibition of cytochrome P450 3A. Concentrations of spectrally detectable cytochrome P450 were decreased after incubation with methyl parathion. The decreased absorbance at 450 run was not associated with comparable increases at 420 nm, suggesting the displacement of heme from the enzyme possibly due to the effects of free sulfur. Data from the analysis of protein-bound heme indicate that heme was lost during the metabolism of methyl parathion. However, mass spectral analysis of free and protein-bound heme did not result in the detection of heme adducts or heme fragments, suggesting the degradation of heme after displacement from the enzyme.; Proteomic analysis of cytochrome P450 3A subsequent to incubation with methyl parathion indicated that the tryptic peptide associated with cysteine #443 within the active site of the enzyme is altered in such a way to prevent its detection. Proteomic analysis also revealed 96 amu adducts to cysteines #67 and #377 in cytochrome P450 3A1. These adducts are likely to be (1) three sulfur atoms, or (2) sulfur and oxygen in combination as S2O 2 or SO2S.