Allosteric effects on substrate dissociation from cytochrome P450 3A4 in nanodiscs observed by ensemble and single-molecule fluorescence spectroscopy.
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.
Nath
Abhinav
A
Department of Medicinal Chemistry, University of Washington, Seattle, Washington, USA.
Koo
Peter K
PK
Rhoades
Elizabeth
E
Atkins
William M
WM
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
D010078:Oxazines; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C044808:nile red