CRYSTALLOGRAPHIC STUDIES ON CYTOCHROME P450

这项提案的目的是继续晶体学 恶臭假单胞菌细胞色素P450cam的研究 我们有 将底物结合的酶完全精制至1.61埃 和无底物酶至2.2埃。 我们现在希望 确定了P450cam与一系列 抑制剂和除樟脑以外的基质， 了解底物特异性的结构基础。 在 此外，我们计划解决的晶体结构的 机械上重要的氧-P450-樟脑三元复合物. 低温 在每一项研究中， 将针对晶体学温度因素作为一种手段， 获取功能上重要的动态信息。 在 与S博士合作斯利格，我们会确定水晶 P450cam位点定向变异体的结构。 作为血红素酶动力学的平行研究，我们将探讨 配体结合对构象和动力学的影响 细胞色素c过氧化物酶的性质。 最后，我们打算解决 至少有一个其他P450的结构，以直接测试的想法，我们 从P450凸轮结构上有哪些因素控制 底物特异性 目前的建议将补充我们的整体研究计划 血红素酶的结构和功能。 具体来说，我们是 研究细胞色素c过氧化物酶的晶体结构- 细胞色素c复合物、辣根过氧化物酶和假单胞菌 铜绿菌细胞色素氧化酶 每一种都是由 过氧化物酶-细胞色素C复合物的研究正在顺利进行。

The purpose of this proposal is to continue crystallographic studies on cytochrome P450cam from Pseudomonas putida. We have completed refinement of the substrate-bound enzyme to 1.61 angstrom and of the substrate-free enzyme to 2.2 angstrom. We now wish to determine the crystal structure of P450cam complexed with a series of inhibitors and substrates other than camphor to better understand the structural basis for substrate specificity. In addition, we plan to solve the crystal structure of the mechanistically important oxy-P450-camphor ternary complex at cryogenic temperatures. In each of ese studies, special attention will be directed to crystallographic temperature factors as a means of obtaining functionally important dynamical information. In collaboration with Dr. S. Sligar, we will determine the crystal structure of site-directed P450cam variants. As a parallel study on heme enzyme dynamics, we will explore the effect of ligand binding on the conformational and dynamical properties of cytochrome c peroxidase. Finally, we intend to solve the structure of at least one other P450 to directly test ideas we have developed from the P450cam structure on what factors control substrate specificity. The present proposal will complement our overall research program on heme enzyme structure and function. Specifically, we are working on the crystal structures of the cytochrome c peroxidase- cytochrome c complex, horseradish peroxidase, and Pseudomonas aerguinosa cytochrome oxidase. Each of these has been crystallized and work on the peroxidase-cytochrome c complex is well underway.