Zhang Haoming, Lin Hsia-lien, Walker Vyvyca J, Hamdane Djemel, Hollenberg Paul F
Department of Pharmacology, The University of Michigan, Ann Arbor, MI 48109-5632, USA.
Mol Pharmacol. 2009 Nov;76(5):1011-8. doi: 10.1124/mol.109.059808. Epub 2009 Aug 31.
We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a K(I) of 0.44 microM and k(inact) of 0.12 min(-1). The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 A away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by approximately 20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4.
我们已经证明，4-（叔丁基）苯乙炔（tBPA）在重组系统中是一种有效的基于机制的细胞色素P450 2B4（P450 2B4）失活剂。它以NADPH和时间依赖性方式使P450 2B4失活，K（I）为0.44 microM，k（inact）为0.12 min⁻¹。分配比约为零，表明失活发生时反应中间体未离开活性位点。液相色谱-质谱分析表明，tBPA以1:1的化学计量比形成蛋白质加合物。对tBPA修饰的蛋白质进行肽图谱分析提供了证据，表明tBPA与Thr302共价结合。这与分子模拟结果一致，该结果显示炔基的末端碳原子距离Thr302仅3.65 Å。为了表征Thr302共价修饰的影响，从重组系统中纯化得到了均一的tBPA修饰的P450 2B4。与未修饰的蛋白质相比，tBPA修饰的蛋白质的Soret带红移至5至422 nm。苯丙胺与修饰的P450 2B4结合不会引起自旋位移，表明底物结合和/或血红素环境已被共价结合的tBPA改变。细胞色素P450还原酶以大致相同的速率还原未修饰的和tBPA修饰的P450。然而，加入苯丙胺可使未修饰的P450 2B4的还原速率提高约20倍，但仅略微刺激tBPA修饰的蛋白质的还原。苯丙胺对第一次电子转移的刺激存在如此大的差异，强烈表明P450催化的受损是由于苯丙胺与tBPA修饰的P450 2B4结合受到抑制。
