title: Rapid conformational dynamics of cytochrome P450 2E1 in a natural biological membrane environment.
abstract: Among the members of the cytochrome P450 superfamily, P450 2E1 is most often associated with the production of reactive oxygen species and subsequent cellular toxicity. We sought to identify a structural basis for this distinguishing feature of P450 2E1 by examining its carbon monoxide binding kinetics as a probe of conformation/dynamics. We employed liver microsomes from wild-type and P450 2E1 knockout mice in order to characterize this P450 in a natural membrane environment. The CO binding kinetics of the P450s of wild-type microsomes had a rapid component that was absent in the knockout microsomes. Data analysis using the maximum entropy method (MEM) correspondingly identified two distinct kinetic components in the wild-type microsomes and only one component in the knockout microsomes. The rapid kinetic component in wild-type microsomes was attributed to endogenous P450 2E1, while the slower component was derived from the remaining P450s. In addition, rapid binding kinetics and a single component were also observed for human P450 2E1 in a baculovirus expression system, in the absence of other P450s. Binding kinetics of both mouse and human P450 2E1 were slowed in the presence of ethanol, a modulator of this P450. The unusually rapid CO binding kinetics of P450 2E1 indicate that it is more dynamically mobile than other P450s and thus able to more readily interconvert among alternate conformations. This suggests that conformational switching during the catalytic cycle may promote substrate release from a short-lived binding site, allowing activated oxygen to attack other targets with toxic consequences.
authors.affiliation: Department of Pharmacology and Toxicology, The University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA. svsmith@pharmacology.umsmed.edu
authors.forename: Stanley V
authors.initials: SV
authors.lastname: Smith
authors.forename: Richard C
authors.initials: RC
authors.lastname: Robinson
authors.forename: Tina G
authors.initials: TG
authors.lastname: Smith
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Burks
authors.forename: Fred K
authors.initials: FK
authors.lastname: Friedman
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D007527:Isoenzymes; D002248:Carbon Monoxide; D019392:Cytochrome P-450 CYP2E1