STUDIES ON THE ACTIVE SITES AND MECHANISMS OF CYTOCHROME P450

尽管已经做出了广泛的努力来确定 细胞色素P450催化循环，这些酶的几个方面 阻碍了他们的完整角色。其中包括缺席 晶体结构的复杂性(对于哺乳动物的酶)， 催化循环，存在多种同工酶，多样性 底物(对于某些同工酶)和活性氧的多功能性 物种。细胞色素P450 2 A1的同位素效应对细胞周期变化的敏感性 还原速率表明这种酶可以用来研究 P450、还原酶和细胞色素b-5之间的相互作用。同位素 细胞色素P450 2 A1对睾酮羟化作用的影响 还原酶水平和细胞色素b-5的存在。尽管这两个人 这些蛋白质被认为与细胞色素的还原有关。 P450，它们对观测的同位素效应有不同的影响。 正如预期的那样，不断增加的还原酶水平会导致 观察到的同位素效应，因为水的形成揭示了P450 同位素效应，依赖于还原速率。然而，在场的人 细胞色素b-5会导致观察到的同位素效应的降低， 这表明这种蛋白质要么干扰了 第三个电子或充当电子接收器，阻止还原 活性氧的含量。 CYP3A家族是人类药物中比较重要的P450家族之一 新陈代谢。不止一种药物的存在，除了引起 抑制，也可以激活这些酶。菲的代谢是 被7，8-苯并黄酮激活，而7，8-苯并黄酮本身就是底物 对于细胞色素P3A4。对这两种底物的动力学分析表明，7，8- 苯并黄酮可增加菲代谢的V-max 改变K-m和菲降低了7，8- 在不增加K-m的情况下苯黄酮类代谢。这些结果 建议两种底物(或底物和活化剂)都是 同时出现在活动站点中。这些数据提供了第一个 两个不同的分子可以同时结合到 相同的P450活性部位。

Although there has been an extensive effort to determine the details of the cytochrome P450 catalytic cycle, several aspects of these enzymes have hindered their complete characterization. These include the absence of crystal structures (for mammalian enzymes), the complexity of the catalytic cycle, presence of multiple isozymes, the diversity of substrates (for some isozymes), and the versatility of the active oxygen species. The sensitivity of CYP2A1 isotope effects to changes in reduction rates suggests that this enzyme could be used to study the interactions between P450s, reductase and cytochrome b-5. The isotope effect for hydroxylation of testosterone by CYP2A1 is influenced by both reductase levels and the presence of cytochrome b-5. Although both of these proteins are thought to be involved in the reduction of cytochrome P450s, they have different influences on the observed isotope effects. As expected, increasing reductase levels causes an increase in the observed isotope effect, since water formation, which unmasks a P450 isotope effect, is dependent on reduction rate. However, the presence of cytochrome b-5 causes a decrease in the observed isotope effect, suggesting that this protein either interferes with the introduction of the third electron or acts as an electron sink, preventing the reduction of the active oxygen. The CYP3A family is one of the more important P450s in human drug metabolism. The presence of more than one drug, in addition to causing inhibition, can also activate these enzymes. Phenanthrene metabolism is activated by 7,8-benzoflavone, and 7,8-benzoflavone is itself a substrate for CYP3A4. Kinetic analyses of these two substrates shows that 7,8- benzoflavone increases the V-max of phenanthrene metabolism without changing the K-m and that phenanthrene decreases the V-max of 7,8- benzoflavone metabolism with out increasing the K-m. These results suggest that both substrates (or substrate and activator) are simultaneously present in the active site. These data provide the first evidence that two different molecules can be bound simultaneously to the same P450 active site.