Effect of 17-α-ethynylestradiol on activities of cytochrome P4502B (P450 2B) enzymes:: Characterization of inactivation of P450s 2B1 and 2B6 and identification of metabolites
Effect of 17-α-ethynylestradiol on activities of cytochrome P4502B (P450 2B) enzymes:: Characterization of inactivation of P450s 2B1 and 2B6 and identification of metabolites
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DOI：
10.1124/jpet.300.2.549
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发表时间：
2002-02-01
影响因子：
3.5
通讯作者：
Hollenberg, PF
中科院分区：
文献类型：
--
作者：
Kent, UM;Mills, DE;Hollenberg, PF
17-α-乙炔雌二醇 (17EE) 以基于机制的方式灭活纯化、重构的大鼠肝细胞色素 P450 (P450) 2B1 和人 P450 2B6。当 P450 2B2 或 2B4 与 17EE 一起孵育时，几乎没有观察到失活。 P450s 2B1 和 2B6 的失活完全依赖于 NADPH 和 17EE，并遵循伪一级动力学。 P450s 2B1和2B6在30℃失活的最大速率常数分别为0.2和0.03min(-1)。对于 P450 2B1 和 2B6，表观 K-1 分别为 11 和 0.8 μM。 P450 2B1 与 17EE 和 NADPH 一起孵育 20 分钟，导致酶活性损失 75%，同时酶形成还原 CO 复合物的能力损失 20% 至 25%。使用 P450 2B6，观察到酶活性损失 83%，CO 还原谱损失 5% 至 10%。 17EE 与 P450 2B1 和 2B6 的外推分配比分别为 21 和 13。同时孵育替代底物与 17EE 可防止两种酶失活。观察到放射性标记的 17EE 与 P450 2B1 和 2136 结合的化学计量为 1.3:1。这些结果表明，17EE 以基于机制的方式灭活 P450 2B1 和 2B6，主要是通过 17EE 的反应中间体与脱辅基蛋白的结合。对 17EE 代谢物的分析表明，失活的 2B 酶的差异主要在于它们产生 P450 2B2 或 2B4 不产生的两种代谢物的能力。
17-alpha-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 30degreesC were 0.2 and 0.03 min(-1), respectively. For P450s 2B1 and 2B6 the apparent K-1 was 11 and 0.8 muM, 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 2136 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.