title: Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment.
abstract: Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.
authors.affiliation: Department of Biochemistry, University of Illinois Urbana-Champaign, 505 South Goodwin Avenue, IL 61801, USA.
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Baas
authors.forename: Ilia G
authors.initials: IG
authors.lastname: Denisov
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Sligar
mesh_terms: D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004347:Drug Interactions; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006900:Hydroxylation; D008051:Lipid Bilayers / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D036103:Nanotechnology; D012995:Solubility; D013057:Spectrum Analysis; D013739:Testosterone / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A