Identification of 17-alpha-ethynylestradiol-modified active site peptides and glutathione conjugates formed during metabolism and inactivation of P450s 2B1 and 2B6.
The oral contraceptive 17-alpha-ethynylestradiol (17EE) is a mechanism-based inactivator of cytochrome P450s (P450s) 2B1 and 2B6. Inactivation of P450s 2B1 and 2B6 in the reconstituted system by [3H]17EE resulted in labeling of the P450 apoprotein. Mass spectral analysis of 17EE-inactivated P450 2B1 showed an increase in the mass of the apoprotein by 313 Da, consistent with the mass of 17EE plus one oxygen atom. P450s 2B1 and 2B6 were inactivated with [3H]17EE and digested with CNBr. Separation of these peptides resulted in the identification of one major labeled peptide for each enzyme. N-Terminal sequencing of these peptides yielded the amino acid sequences PYTDAVIHEI (for P450 2B1) and PYTEAV (for P450 2B6) that corresponded to amino acids P347-M376 and P347-M365 in P450s 2B1 and 2B6, respectively. Electrospray ionization (ESI)-liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization (MALDI)-MS analysis of the P450 2B1-derived peptide resulted in a mass of 3654 Da consistent with the mass of the P347-M376 peptide (3385 Da) plus a 268 Da 17EE adduct. Chemically reactive intermediates of 17EE that were generated during the metabolism of 17EE by P450s 2B1 and 2B6 were trapped with gluthathione (GSH). ESI-LC-MS/MS analysis of 17EE-GSH conjugates from the incubation mixtures indicated that P450s 2B1 and 2B6 generated different reactive 17EE intermediates that were responsible for the inactivation and protein modification or the formation of GSH conjugates by these two enzymes.
Department of Pharmacology, The University of Michigan, Medical Science Research Building III, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USA.
Ute M
UM
Kent
Hsia-Lien
HL
Lin
Danielle E
DE
Mills
Kelly A
KA
Regal
Paul F
PF
Hollenberg
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019362:Cytochrome P-450 CYP2B1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D065702:Cytochrome P-450 CYP2B6; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004997:Ethinyl Estradiol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D010455:Peptides / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D020318:Rats, Long-Evans; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013329:Structure-Activity Relationship
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D010455:Peptides; D004997:Ethinyl Estradiol; D003577:Cytochrome P-450 Enzyme System; C585599:CYP2B6 protein, human; D019362:Cytochrome P-450 CYP2B1; D065702:Cytochrome P-450 CYP2B6; D005978:Glutathione