title: Subcellular localization of rat CYP2E1 impacts metabolic efficiency toward common substrates.
abstract: Cytochrome P450 2E1 (CYP2E1) detoxifies or bioactivates many low molecular-weight compounds. Most knowledge about CYP2E1 activity relies on studies of the enzyme localized to endoplasmic reticulum (erCYP2E1); however, CYP2E1 undergoes transport to mitochondria (mtCYP2E1) and becomes metabolically active. We report the first comparison of in vitro steady-state kinetic profiles for erCYP2E1 and mtCYP2E1 oxidation of probe substrate 4-nitrophenol and pollutants styrene and aniline using subcellular fractions from rat liver. For all substrates, metabolic efficiency changed with substrate concentration for erCYP2E1 reflected in non-hyperbolic kinetic profiles but not for mtCYP2E1. Hyperbolic kinetic profiles for the mitochondrial enzyme were consistent with Michaelis-Menten mechanism in which metabolic efficiency was constant. By contrast, erCYP2E1 metabolism of 4-nitrophenol led to a loss of enzyme efficiency at high substrate concentrations when substrate inhibited the reaction. Similarly, aniline metabolism by erCYP2E1 demonstrated negative cooperativity as metabolic efficiency decreased with increasing substrate concentration. The opposite was observed for erCYP2E1 oxidation of styrene; the sigmoidal kinetic profile indicated increased efficiency at higher substrate concentrations. These mechanisms and CYP2E1 levels in mitochondria and endoplasmic reticulum were used to estimate the impact of CYP2E1 subcellular localization on metabolic flux of pollutants. Those models showed that erCYP2E1 mainly carries out aniline metabolism at all aniline concentrations. Conversely, mtCYP2E1 dominates styrene oxidation at low styrene concentrations and erCYP2E1 at higher concentrations. Taken together, subcellular localization of CYP2E1 results in distinctly different enzyme activities that could impact overall metabolic clearance and/or activation of substrates and thus impact the interpretation and prediction of toxicological outcomes.
authors.affiliation: Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, United States.
authors.forename: Jessica H
authors.initials: JH
authors.lastname: Hartman
authors.affiliation: Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, United States; Department of Chemistry, University of Central Arkansas, Conway, AR, United States.
authors.forename: H Cass
authors.initials: HC
authors.lastname: Martin
authors.affiliation: Department of Chemistry, Hendrix College, Conway, AR, United States.
authors.forename: Andres A
authors.initials: AA
authors.lastname: Caro
authors.affiliation: Arkansas Biosciences Institute, United States; Psychology & Counseling, Arkansas State University, Jonesboro, AR, United States.
authors.forename: Amy R
authors.initials: AR
authors.lastname: Pearce
authors.affiliation: Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, United States. Electronic address: MillerGroverP@uams.edu.
authors.forename: Grover P
authors.initials: GP
authors.lastname: Miller
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D001711:Biotransformation; D020134:Catalytic Domain; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005260:Female; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D008954:Models, Biological; D009596:Nitrophenols / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D021381:Protein Transport; D017207:Rats, Sprague-Dawley; D020058:Styrene / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000814:Aniline Compounds; D009596:Nitrophenols; D020058:Styrene; D019392:Cytochrome P-450 CYP2E1; C023650:aniline; C024836:4-nitrophenol
keywords: 4-Nitrophenol; Aniline; CYP2E1; Cooperativity; Mitochondrial transport; Styrene