Mechanism-based inactivation of cytochrome P450 3A4 by 17α-ethynylestradiol:: Evidence for heme destruction and covalent binding to protein
Mechanism-based inactivation of cytochrome P450 3A4 by 17α-ethynylestradiol:: Evidence for heme destruction and covalent binding to protein
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DOI：
10.1124/jpet.301.1.160
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发表时间：
2002-04-01
影响因子：
3.5
通讯作者：
Hollenberg, PF
中科院分区：
文献类型：
--
作者：
Lin, HL;Kent, UM;Hollenberg, PF
17 α -乙炔雌醇（EE）是许多口服避孕药的主要成分，它以一种基于机制的方式使用磷脂和nadph -细胞色素P450还原酶重组的纯化P450 3A4的睾酮6 - β羟基化活性失活。P450 3A4的失活遵循准一级动力学，并依赖于NADPH。k - i和k（原状）值分别为18 muM和0.04 min(-1), t（1/2）为16 min。50个muM EE与P450 3A4在37°c下孵育30分钟，导致睾酮6 -羟化活性降低67%，原生蛋白在415 nm处的光谱吸收度降低35%，光谱可检测的P450- co复合物损失70%。EE对P450 3A4的失活是不可逆的。睾酮，一种替代底物，能够保护P450 3A4免受ee依赖性失活。分割比近似为50。结合的化学计量是每nmol P450 3A4失活约1.3 nmol EE代谢物结合。sds -聚丙烯酰胺凝胶电泳分析表明[H-3] EE与P450 3A4载脂蛋白不可逆结合。在对[H-3] EE失活样品进行大量透析后，高压液相色谱（HPLC）分析表明，EE代谢导致的失活导致大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载脂蛋白。电喷雾质谱分析表明，与EE代谢的主要放射性标记产物相对应的部分质量（M - H）（-）为479 Da。HPLC和气相色谱-质谱分析显示，P450 3A4代谢EE产生一个主要代谢物，2-羟乙基雌二醇，以及至少三个额外的代谢物。综上所述，我们的研究结果表明，EE是一种有效的基于机制的P450 3A4失活剂，其失活机制不仅涉及血红素破坏，还涉及活性位点载脂蛋白的不可逆修饰。
17alpha-Ethynylestradiol (EE), a major constituent of many oral contraceptives, inactivated the testosterone 6beta-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 muM and 0.04 min(-1), respectively, and the t(1/2) was 16 min. Incubation of 50 muM EE with P450 3A4 at 37degreesC for 30 min resulted in a 67% loss of testosterone 6beta-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 similar to50. 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 [H-3] EE was irreversibly bound to the P450 3A4 apoprotein. After extensive dialysis of the [H-3] 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.