STUDIES ON THE ACTIVE SITES AND MECHANISMS OF CYTOCHROME P450

点击翻译按钮获取中文摘要

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.