title: Probing the CYP3A4 active site by cysteine scanning mutagenesis and photoaffinity labeling.
abstract: The mechanism of CYP3A4-substrate interactions has been investigated using a battery of techniques including cysteine scanning mutagenesis, photoaffinity labeling, and structural modeling. In this study, cysteine scanning mutagenesis was performed at seven sites within CYP3A4 proposed to be involved in substrate interaction and/or cooperativity. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis for each mutant after proteolytic digestion and isolation of fluorescent photolabeled peptides. Among the tryptic peptides of seven tested mutants, three photolabeled peptides of the F108C mutant, ECYSVFTNR (positions 97-105), VLQNFSFKPCK (positions 459-469), and RPCGPVGFMK (positions 106-115) were identified by MALDI-TOF-MS and nano-LC/ESI QTOF MS. The site of modification was further localized to the substituted Cys-108 residue in the mutant peptide adduct RPCGPVGFMK (positions 106-115) by nano-LC/ESI QTOF MS/MS. In summary, we described a potentially useful method to study P450 active sites using a combination of cysteine scanning mutagenesis and photoaffinity labeling.
authors.affiliation: Department of Medicinal Chemistry, Box 357610, University of Washington, Seattle, WA 98195, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Wen
authors.forename: Catalin E
authors.initials: CE
authors.lastname: Doneanu
authors.forename: Jed N
authors.initials: JN
authors.lastname: Lampe
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Roberts
authors.forename: William M
authors.initials: WM
authors.lastname: Atkins
authors.forename: Sidney D
authors.initials: SD
authors.lastname: Nelson
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003545:Cysteine / Q000032:analysis / Q000737:chemistry*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation; D015336:Molecular Probe Techniques; D016297:Mutagenesis, Site-Directed; D019920:Photoaffinity Labels / Q000032:analysis / Q000737:chemistry*; D011485:Protein Binding; D013329:Structure-Activity Relationship
chemical_list: D019920:Photoaffinity Labels; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D003545:Cysteine