17-␣-Ethynylestradiol (17EE) inactivated purified, reconstituted rat hepatic cytochrome P450 (P450) 2B1 and human P450 2B6 in a mechanism-based manner. Little or no inactivation was observed when P450s 2B2 or 2B4 were incubated with 17EE. The inactivation of P450s 2B1 and 2B6 was entirely dependent on both NADPH and 17EE and followed pseudo-first order kinetics. The maximal rate constants for the inactivation of P450s 2B1 and 2B6 at 30°C were 0.2 and 0.03 min Ϫ1 , respectively. For P450s 2B1 and 2B6 the apparent K I was 11 and 0.8 M, respectively. Incubation of P450 2B1 with 17EE and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 20 to 25% loss of the enzyme's ability to form a reduced CO complex. With P450 2B6, an 83% loss in enzymatic activity and a 5 to 10% loss in the CO reduced spectrum were observed. The extrapolated partition ratios for 17EE with P450 2B1 and 2B6 were 21 and 13, respectively. Simultaneous incubation of an alternate substrate together with 17EE protected both enzymes from inactivation. A 1.3:1 stoichiometry of labeling for binding of the radiolabeled 17EE to P450 2B1 and 2B6 was seen. These results indicate that 17EE inactivates P450s 2B1 and 2B6 in a mechanism-based manner, primarily by the binding of a reactive intermediate of 17EE to the apoprotein. Analysis of the 17EE metabolites showed that 2B enzymes that become inactivated differ primarily by their ability to generate two metabolites that were not produced by P450s 2B2 or 2B4.

The oral contraceptive 17-α-ethynylestradiol (17EE1) is a mechanism-based inactivator of P450s 2B1 and 2B6. Inactivation of P450s 2B1 and 2B6 in the reconstituted system by [ 3 H]17EE resulted in labeling of the P450 apo-protein. 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 [ 3 H]17EE and digested with CNBr. Separation of the 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 P 347 -M 376 and P 347 -M 365 in P450s 2B1 and 2B6, respectively. ESI-LC-MS and MALDI-TOF-MS analysis of the P450 2B1-derived peptide resulted in a mass of 3654 Da consistent with the mass of the P 347 -M 376 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.The P450 enzymes belong to a family of microsomal cytochromes that are characterized by their unique heme absorbance spectrum at 450 nm in the reduced, carbon monoxide bound form (1). Mammalian P450s play a pivotal role in the detoxification, metabolism, and clearance of the majority of drugs as well as many carcinogens and pesticides (2). In many instances metabolism of xenobiotics by P450 enzymes results in the production of reactive intermediates. The formation of reactive intermediates that bind to cellular proteins or DNA has been implicated in the toxic or carcinogenic effects of certain environmental pollutants and drugs. Recently, it has also been suggested that protein adduction by reactive intermediates plays a significant role in the mechanisms of some neurotoxic events (3).The membrane-bound nature of mammalian P450s has made the characterization of their active sites and the elucidation of their three-dimensional structures particularly difficult. Although several crystal structures of modified P450s have been solved (4-8) much of the current 1 Abbreviations: P450, cytochrome P450; Reductase, NADPH-cytochrome P450 reductase; DLPC, dilauroyl-L-α-phosphatidylcholine; 17EE, 17-α-ethynylestradiol; BSA, bovine serum albumin; 7-EFC, 7-ethoxy-4-trifluoromethyl)coumarin; HFC, 7-hydroxy-4-(trifluoromethyl)coumarin; LC-MS, liquid chromatography-mass spectrometry; ESI, electrospray ionization; MALDI, matrix-assisted laser desorption ionization. *To whom correspondence should be addressed at the Department of Pharmacology, Medical Science Research Bldg. III, 1150 West Medical Center Dr., Ann Arbor, MI 48109...