CRYSTALLOGRAPHIC STUDIES ON CYTOCHROME P450

这项建议的目的是继续结晶学。 恶臭假单胞菌细胞色素P450 cam的研究我们有 底物结合酶完全提纯至1.61埃 无底物酶活力为2.2埃。我们现在希望 一系列络合物P450 cam的晶体结构测定 除樟脑外的其他抑制剂和底物，以更好地 了解底物专一性的结构基础。在……里面 此外，我们计划解决晶体结构的问题 力学上重要的氧基-P450-樟脑三元络合物 低温温度。在每一项ESE研究中，都特别注意 将以晶体温度因素为手段 获得功能上重要的动力学信息。在……里面 与S·斯莱格博士合作，我们将确定晶体 位点定向的P450 cam变体的结构。 作为对血红素酶动力学的平行研究，我们将探索 配基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的结构直接测试我们的想法 由P450凸轮结构发展而来的对哪些因素的控制 底物专一性。 这项建议将补充我们的整体研究计划 关于血红素酶的结构和功能。具体地说，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复合体、辣根过氧化物酶和假单胞菌 铜绿假单胞菌细胞色素氧化酶。其中的每一个都被结晶了 过氧化物酶-细胞色素c复合体的研究进展顺利。

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.