Lin Hsia-lien, Kent Ute M, Hollenberg Paul F
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-0632, USA.
J Pharmacol Exp Ther. 2002 Apr;301(1):160-7. doi: 10.1124/jpet.301.1.160.
17 alpha-Ethynylestradiol (EE), a major constituent of many oral contraceptives, inactivated the testosterone 6 beta-hydroxylation activity of purified P450 3A4 reconstituted with phospholipid and NADPH-cytochrome P450 reductase in a mechanism-based manner. The inactivation of P450 3A4 followed pseudo first order kinetics and was dependent on NADPH. The values for the K(I) and k(inact) were 18 microM and 0.04 min(-1), respectively, and the t(1/2) was 16 min. Incubation of 50 microM EE with P450 3A4 at 37 degrees C for 30 min resulted in a 67% loss of testosterone 6 beta-hydroxylation activity accompanied by a 35% loss of the spectral absorbance of the native protein at 415 nm and a 70% loss of the spectrally detectable P450-CO complex. The inactivation of P450 3A4 by EE was irreversible. Testosterone, an alternate substrate, was able to protect P450 3A4 from EE-dependent inactivation. The partition ratio was approximately 50. The stoichiometry of binding was approximately 1.3 nmol of an EE metabolite bound per nmol of P450 3A4 inactivated. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [(3)H]EE was irreversibly bound to the P450 3A4 apoprotein. After extensive dialysis of the [(3)H]EE inactivated samples, high-pressure liquid chromatography (HPLC) analysis demonstrated that the inactivation resulting from EE metabolism led to the destruction of approximately half the heme with the concomitant generation of modified heme and EE-labeled heme fragments and produced covalently radiolabeled P450 3A4 apoprotein. Electrospray mass spectrometry demonstrated that the fraction corresponding to the major radiolabeled product of EE metabolism has a mass (M - H)(-) of 479 Da. HPLC and gas chromatography-mass spectometry analyses revealed that EE metabolism by P450 3A4 generated one major metabolite, 2-hydroxyethynylestradiol, and at least three additional metabolites. In conclusion, our results demonstrate that EE is an effective mechanism-based inactivator of P450 3A4 and that the mechanism of inactivation involves not only heme destruction, but also the irreversible modification of the apoprotein at the active site.
17α-乙炔雌二醇（EE）是许多口服避孕药的主要成分，它以基于机制的方式使与磷脂和NADPH-细胞色素P450还原酶重构的纯化P450 3A4的睾酮6β-羟基化活性失活。P450 3A4的失活遵循伪一级动力学，且依赖于NADPH。K（I）和k（inact）的值分别为18μM和0.04 min⁻¹，t（1/2）为16分钟。在37℃下将50μM EE与P450 3A4孵育30分钟，导致睾酮6β-羟基化活性丧失67%，同时天然蛋白质在415nm处的光谱吸光度丧失35%，光谱可检测的P450-CO复合物丧失70%。EE对P450 3A4的失活是不可逆的。睾酮作为替代底物，能够保护P450 3A4免受EE依赖性失活。分配比约为50。结合化学计量约为每失活1 nmol P450 3A4结合1.3 nmol的EE代谢物。SDS-聚丙烯酰胺凝胶电泳分析表明，[³H]EE与P450 3A4脱辅基蛋白不可逆结合。对[³H]EE失活样品进行广泛透析后，高压液相色谱（HPLC）分析表明，EE代谢导致的失活导致约一半的血红素被破坏，同时产生修饰的血红素和EE标记的血红素片段，并产生共价放射性标记的P450 3A4脱辅基蛋白。电喷雾质谱表明，与EE代谢的主要放射性标记产物相对应的馏分的质量（M - H）⁻为479 Da。HPLC和气相色谱-质谱分析表明，P450 3A4对EE的代谢产生一种主要代谢物2-羟基乙炔雌二醇，以及至少三种其他代谢物。总之，我们的结果表明，EE是一种有效的基于机制的P450 3A4失活剂，失活机制不仅涉及血红素破坏，还涉及活性位点脱辅基蛋白的不可逆修饰。
