title: CYP2E1 hydroxylation of aniline involves negative cooperativity.
abstract: CYP2E1 plays a role in the metabolic activation and elimination of aniline, yet there are conflicting reports on its mechanism of action, and hence relevance, in aniline metabolism. Based on our work with similar compounds, we hypothesized that aniline binds two CYP2E1 sites during metabolism resulting in cooperative reaction kinetics and tested this hypothesis through rigorous in vitro studies. The kinetic profile for recombinant CYP2E1 demonstrated significant negative cooperativity based on a fit of data to the Hill equation (n=0.56). Mechanistically, the data were best explained through a two-binding site cooperative model in which aniline binds with high affinity (K(s)=30 μM) followed by a second weaker binding event (K(ss)=1100 uM) resulting in a threefold increase in the oxidation rate. Binding sites for aniline were confirmed by inhibition studies with 4-methylpyrazole. Inhibitor phenotyping experiments with human liver microsomes validated the central role for CYP2E1 in aniline hydroxylation and indicated minor roles for CYP2A6 and CYP2C9. Importantly, inhibition of minor metabolic pathways resulted in a kinetic profile for microsomal CYP2E1 that replicated the preferred mechanism and parameters observed with the recombinant enzyme. Scaled modeling of in vitro CYP2E1 metabolism of aniline to in vivo clearance, especially at low aniline levels, led to significant deviations from the traditional model based on non-cooperative, Michaelis-Menten kinetics. These findings provide a critical mechanistic perspective on the potential importance of CYP2E1 in the metabolic activation and elimination of aniline as well as the first experimental evidence of a negatively cooperative metabolic reaction catalyzed by CYP2E1.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Jessica H
authors.initials: JH
authors.lastname: Hartman
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA; Department of Chemistry, University of Central Arkansas, Conway, AR 72205, USA.
authors.forename: Katie
authors.initials: K
authors.lastname: Knott
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. Electronic address: MillerGroverP@uams.edu.
authors.forename: Grover P
authors.initials: GP
authors.lastname: Miller
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism*; D000077604:Fomepizole; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D006900:Hydroxylation; D008862:Microsomes, Liver / Q000201:enzymology; D011720:Pyrazoles
chemical_list: D000814:Aniline Compounds; D011720:Pyrazoles; D000077604:Fomepizole; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; C023650:aniline
keywords: Aniline; CYP2E1; Cooperativity; Cytochrome P450; HPLC; Metabolism; high performance liquid chromatography