Effect of 17-#alpha#-Ethynylestradiol on Activities of Cytochrome P450 2B (P450 2B) Enzymes: Characterization of Inactivation of P450s 2B1 and 2B6 and Identification of Metaoblites

摘要

17-#alpha#-Ethynylestradiol (17EE) inactivated purified, reconstituted rat hepatic cytochrome P450 (450) 2B1 and human P450 2B6 in a mechanism-based manner. Little or no inactivation was observed when P450s 2B2 or 2B4 were inclubated 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 deg C were 0.2 and 0.03 min~(-1), respectively. For P450s 2B1 and 2B6 the apparent K_I was 11 and 0.8 #mu#M, respectively. Incubation 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 1.1:1 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 metaoblites showed that 2B enzymes that become inactivated differ primarily by their ability to generate two metaoblites that were not produced by P450s 2B2 or 2B4.