title: Mass spectrometric detection of CYP450 adducts following oxidative desulfuration of methyl parathion.
abstract: Cytochrome P450 (CYP)-mediated desulfuration of methyl parathion results in mechanism-based inhibition of the enzyme. Although previous data suggest that reactive sulfur is released and binds to the apoprotein, the identities of neither the adduct(s) nor the affected amino acid(s) have been clearly determined. In this study, nanospray tandem mass spectroscopy was used to analyze peptide digests of CYP resolved by SDS-PAGE from liver microsomes of male rats following incubation in the absence or presence of methyl parathion. Oxidative desulfuration was confirmed by measurement of methyl paraoxon, and inhibition of specific CYP isozymes was determined by measurement of testosterone hydroxylation. Total CYP content was quantified spectrophotometrically. Incubation of microsomes with methyl parathion decreased CYP content by 58%. This effect was not associated with a comparable increase in absorbance at 420 nm, suggesting the displacement of heme from the apoprotein. Rates of testosterone 2β- and 6β-hydroxylation, respectively, were reduced to 8 and 2%, implicating CYP3A and CYP2C11 in the oxidative desulfuration of methyl parathion. Mass spectrometric analysis identified 96 amu adducts to cysteines 64 and 378 of CYP3A1. In addition, a peptide containing cysteine 433 that coordinates with heme was possibly modified as it was detected in control, but not methyl parathion samples. A comparison of rat CYP3A1 with human CYP3A4 suggests that cysteines 64 and 378 reside along the substrate channel, remote from the active site. Alteration of these residues might modulate substrate entry to the binding pocket of the enzyme.
authors.affiliation: Department of Pathology, University of Mississippi Medical Center, Jackson, Mississippi, USA. pkyle@umc.edu
authors.forename: Patrick B
authors.initials: PB
authors.lastname: Kyle
authors.forename: Stanley V
authors.initials: SV
authors.lastname: Smith
authors.forename: Rodney C
authors.initials: RC
authors.lastname: Baker
authors.forename: Robert E
authors.initials: RE
authors.lastname: Kramer
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006900:Hydroxylation; D007306:Insecticides / Q000378:metabolism* / Q000633:toxicity; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008297:Male; D013058:Mass Spectrometry; D008743:Methyl Parathion / Q000378:metabolism* / Q000633:toxicity; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D008958:Models, Molecular; D010084:Oxidation-Reduction; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
chemical_list: D007306:Insecticides; D007527:Isoenzymes; D013739:Testosterone; D008743:Methyl Parathion; D013455:Sulfur; D003577:Cytochrome P-450 Enzyme System