STRUCTURE-FUNCTION OF CYTOCHROME P-450

本项目的重点是结构-功能的表征 细胞色素P450的关系。 大鼠的交联研究 肝微粒体显示，P450 c特异性与 P450 2a和P450还原酶。 苯并[a]芘（BP）代谢数据表明， P450 2a进一步代谢产生的苯酚代谢物之一， P450C。 这些结果支持膜簇模型，其中 P450和P450还原酶作为稳定的复合物存在，而不是作为 单体。 通过免疫荧光法检测BP与纯化的P450 c的结合。 荧光能量转移和偏振技术。 结果 表明还原酶的作用超越了电子 供体，因为它也增强BP与P450 c的结合。 单克隆抗体 (MAb)1-7-1抑制还原酶介导的变化，这表明结合 在P450 c的还原酶受体区域附近。 的热力学研究 甲基非他明与微粒体P450 b的相互作用表明， 4-7-1改变了底物结合的P450自旋平衡，但没有改变 无底物P450 b，在200 ℃附近发生膜相变 改变苄非他明与P450 b的结合。 三维模型 哺乳动物P450的研究正在使用理论和 实验方法。 后者包括识别暴露的 通过蛋白酶消化实验，交联 实验，以确定P450内的空间接近区域 一级序列，以及合成P450肽的抗体用于 鉴定功能上重要序列。 膜形貌研究 涉及微粒体P450 c，P450 b和P450 j，揭示了蛋白水解， 所有三个P450的共同敏感区域。 该地区已被确认为- 并且对应于相对暴露区域中的预测转弯 P450表面 这一发现与一个概念是一致的。 不同种类哺乳动物P450的共同三级结构。 实验正在进行中，以评估是否结构完整的 该区域对于底物结合和活性是必需的。

The focus of this project is the characterization of structure-function relationships for the cytochromes P450. A cross-linking study of rat liver microsomes revealed that P450c is specifically associated with both P450 2a and P450 reductase. Benzo[a]pyrene (BP) metabolism data indicate that P450 2a further metabolizes one of the phenol metabolites generated by P450c. These results support the membrane cluster model in which P450s and P450 reductase exist as stable complexes rather than as monomers. The binding of BP to purified P450c was examined by fluorescence energy transfer and polarization techniques. The results showed that the role of reductase extends beyond that of an electron donor since it also enhances binding of BP to P450c. Monoclonal antibody (MAb) 1-7-1 inhibited reductase-mediated changes, which suggests binding near the reductase receptor region of P450c. A thermodynamic study of the interaction of benzphetamine with microsomal P450b revealed that MAb 4-7-1 alters the P450 spin equilibrium of substrate-bound but not substrate-free P450b, and that a membrane phase transition near 200C alters the binding of benzphetamine to P450b. A three dimensional model of mammalian P450 is being developed using both theoretical and experimental approaches. The latter includes identification of exposed surface regions on P450s by protease digestion experiments, cross-linking experiments to identify spatially proximate regions within the P450 primary sequence, and the use of antibodies to synthetic P450 peptides to identify functionally significant sequences. A membrane topography study involving microsomal P450c, P450b, and P450j revealed a proteolytically sensitive region common to all three P450s. This region has been identi- fied and corresponds to a predicted turn in a relatively exposed region of the P450 surface. This finding is consistent with the concept of a common tertiary structure for different classes of mammalian P450s. Experiments are underway to evaluate whether the structural integrity of this region is essential for substrate binding and activity.