title: Allosteric effects on substrate dissociation from cytochrome P450 3A4 in nanodiscs observed by ensemble and single-molecule fluorescence spectroscopy.
abstract: Cytochrome P450 (CYP) 3A4 is a major human drug-metabolizing enzyme and displays pharmacologically relevant allosteric kinetics caused by multiple substrate and/or effector binding. Here, in the first single-molecule (SM) fluorescence studies of CYPs, we use total internal reflection fluorescence microscopy to measure residence times of the fluorescent dye Nile Red in CYP3A4 incorporated in surface-immobilized lipid Nanodiscs, with and without the effector alpha-naphthoflavone. We find direct evidence that CYP3A4 effectors can decrease substrate off-rates, providing a possible mechanism for effector-mediated enhancement of substrate metabolism. These interesting results highlight the potential of SM methods in studies of CYP allosteric mechanisms.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Abhinav
authors.initials: A
authors.lastname: Nath
authors.forename: Peter K
authors.initials: PK
authors.lastname: Koo
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Rhoades
authors.forename: William M
authors.initials: WM
authors.lastname: Atkins
mesh_terms: D000494:Allosteric Regulation; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D049329:Nanostructures / Q000737:chemistry*; D010078:Oxazines; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D010078:Oxazines; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C044808:nile red