title: Conformational modulation of human cytochrome P450 2E1 by ethanol and other substrates: a CO flash photolysis study.
abstract: The alcohol-inducible cytochrome P450 2E1 is a major human hepatic P450 which metabolizes a broad array of endogenous and exogenous compounds, including ethanol, low-molecular weight toxins, and fatty acids. Several substrates are known to stabilize this P450 and inhibit its cellular degradation. Furthermore, ethanol is a known modulator of P450 2E1 substrate metabolism. We examined the CO binding kinetics of P450 2E1 after laser flash photolysis of the heme-CO bond, to probe the effects of ethanol and other substrates on protein conformation and dynamics. Ethanol had an effect on the two kinetic parameters that describe CO binding: it decreased the rate of CO binding, suggesting a decrease in the protein's conformational flexibility, and increased the photosensitivity, which indicates a local effect in the active site region such as strengthening of the heme-CO bond. Other substrates decreased the CO binding rate to varying degrees. Of particular interest is the effect of arachidonic acid, which abolished photodissociation in the absence of ethanol but had no effect in the presence of ethanol. These results are consistent with a model of P450 2E1 whereby arachidonic acid binds along a long hydrophobic binding pocket and blocks exit of CO from the heme region.
authors.affiliation: Laboratory of Molecular Carcinogenesis, National Cancer Institute, and Bioengineering and Physical Science Program, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: S V
authors.initials: SV
authors.lastname: Smith
authors.forename: A P
authors.initials: AP
authors.lastname: Koley
authors.forename: R
authors.initials: R
authors.lastname: Dai
authors.forename: R C
authors.initials: RC
authors.lastname: Robinson
authors.forename: H
authors.initials: H
authors.lastname: Leong
authors.forename: A
authors.initials: A
authors.lastname: Markowitz
authors.forename: F K
authors.initials: FK
authors.lastname: Friedman
mesh_terms: D016718:Arachidonic Acid / Q000737:chemistry; D001665:Binding Sites / Q000187:drug effects; D002248:Carbon Monoxide / Q000737:chemistry* / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry* / Q000378:metabolism; D000431:Ethanol / Q000737:chemistry*; D006221:Halothane / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D010782:Photolysis / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D008024:Ligands; D016718:Arachidonic Acid; D000431:Ethanol; D002248:Carbon Monoxide; D019392:Cytochrome P-450 CYP2E1; D006221:Halothane