title: Identification of the residue in human CYP3A4 that is covalently modified by bergamottin and the reactive intermediate that contributes to the grapefruit juice effect.
abstract: Previous studies have demonstrated that bergamottin (BG), a component of grapefruit juice, is a mechanism-based inactivator of CYP3A4 and contributes, in part, to the grapefruit juice-drug interaction. Although the covalent binding of [(14)C]BG to the CYP3A4 apoprotein has been demonstrated by SDS-polyacrylamide gel electrophoresis, the identity of the modified amino acid residue and the reactive intermediate species of BG responsible for the inactivation have not been reported. In the present study, we show that inactivation of CYP3A4 by BG results in formation of a modified apoprotein-3A4 and a GSH conjugate, both exhibiting mass increases of 388 Da, which corresponds to the mass of 6',7'-dihydroxybergamottin (DHBG), a metabolite of BG, plus one oxygen atom. To identify the adducted residue, BG-inactivated 3A4 was digested with trypsin, and the digests were then analyzed by liquid chromatography-tandem mass spectrometry (MS/MS). A mass shift of 388 Da was used for the SEQUEST database search, which revealed a mass increase of 388 Da for the peptide with the sequence (272)LQLMIDSQNSK(282), and MS/MS analysis of the adducted peptide demonstrated that Gln273 is the residue modified. Mutagenesis studies showed that the Gln273 to Val mutant was resistant to inactivation by BG and DHBG and did not generate two of the major metabolites of BG formed by 3A4 wild type. In conclusion, we have determined that the reactive intermediate, oxygenated DHBG, covalently binds to Gln273 and thereby contributes to the mechanism-based inactivation of CYP3A4 by BG.
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-5632, USA.
authors.forename: Hsia-Lien
authors.initials: HL
authors.lastname: Lin
authors.forename: Cesar
authors.initials: C
authors.lastname: Kenaan
authors.forename: Paul F
authors.initials: PF
authors.lastname: Hollenberg
mesh_terms: D019943:Amino Acid Substitution; D001051:Apoenzymes / Q000378:metabolism; D001628:Beverages; D001665:Binding Sites; D002853:Chromatography, Liquid; D032083:Citrus paradisi; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000235:genetics; D065692:Cytochrome P-450 CYP3A Inhibitors; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D018565:Food-Drug Interactions; D011564:Furocoumarins / Q000378:metabolism* / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D001051:Apoenzymes; D065692:Cytochrome P-450 CYP3A Inhibitors; D011564:Furocoumarins; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D005978:Glutathione; C068337:bergamottin