Prostaglandin 19-& 20-Hydroxylation by Cytochrome P450

描述(由申请人提供)：这项研究提案将集中于兔细胞色素P450的CYP4A基因亚家族的四个成员的分子和动力学性质的测定，这些成员主要或仅在omega位置上羟化中等和/或长链脂肪酸和二十烷类化合物。细胞色素P4504A4、4A5、4A6和4A7在氨基酸水平上有85%的序列同源性，但它们的底物特异性差异很大。这些酶的生理功能尚不清楚，但大量文献证明它们通过控制血管张力来调节血流动力学。 例如，脑微血管和肾脏微血管被浓度为10-10M的欧米伽-羟基花生四烯酸收缩。在之前的资助期间，这些酶被克隆并在大肠杆菌中使用每种异构体独有的技术进行表达。现在有可能为实验准备足够的量，从而了解它们的功能。这些亲缘关系密切的家族成员的产生使得它们之间可以进行氨基酸差异的比较，这可以解释它们的底物偏好。此外，一个主要的兴趣是确定细胞色素b5与这些酶的相互作用，这导致显著的欧米伽羟化活性的激活。为了了解这些相互作用，提出了以下具体目标：1)CYP4A与底物的相互作用：使用上述表达系统，将构建各种CYP4A的额外突变，以比较唯一的、同源的，在某些情况下相同的残基对所产生的蛋白质活性的作用。在细胞色素P450还原酶和/或细胞色素b5没有和存在的情况下，用停流分光光度法检测不同底物与细胞色素4A的结合常数和结合速率。2)CyP4A与其他蛋白质的相互作用：表面等离子体共振(Biacore)、停流光谱、Soret CD和分析超速离心方法将被用来研究在有或没有细胞色素b5的情况下，各种表达的CyP4A亚家族成员与它们共同的氧化还原伙伴NADPH-细胞色素P450还原酶结合的速率和热力学。此外，CYP4A亚家族成员表面残基的突变将试图绘制这些蛋白质的结合位点。3)细胞色素P4A单加氧酶系统的电子传递/受体特性：在黄素或血红素假体基团的特定波长下，用停流分光光度法测量电子通量NADPH-细胞色素P450还原酶和细胞色素P4As的速率，以确定使用不同底物对细胞色素b5电子传递的影响，以针对每个细胞色素4A。

DESCRIPTION (provided by applicant): This research proposal will focus on the determination of molecular and kinetic properties of four members of the CYP4A gene subfamily of rabbit cytochromes P450 that hydroxylate medium and/or long chain fatty acids and eIcosanoids primarily or exclusively in the omega-position. Cytochromes P4504A4, 4A5, 4A6 and 4A7 share 85% sequence identity at the amino acid level, yet their substrate specificities vary greatly. The physiological functions of these enzymes remain unclear but abundant literature documents their roles in hemodynamic regulation by controlling vascular tone. Cerebral and renal microvessels are contracted by omega-hydroxyarachidonic acid at concentrations of <10-10 M, for example. In the previous funding period these enzymes were cloned and expressed in E. coli using techniques unique to each isoform. It is now possible to prepare adequate quantities for experiments leading to the understanding of their functions. The generation of these closely related family members has permitted comparisons among them with respect to amino acid differences that could account for their substrate preferences. Also, a primary interest is the determination of the interactions of cytochrome b5 with these enzymes, which result in the marked activation of omega-hydroxylation activities. In order to understand these interactions, the following Specific Aims are proposed: 1) interactions of CYP4As with Substrate: Using the aforementioned expression systems, additional mutations of the various CYP4As wilt be constructed to compare the roles of unique, homologous and, in some cases, identical residues on the activities of the resulting proteins with respect to substrate specificity. The binding constants and rates of binding of various substrates to the CYP4As will be examined by stopped-flow spectrophotometry in the absence and presence of NADPHcytochrome P450 reductase and/or cytochrome b5. 2) Interactions of CYP4As with Other Proteins: Surface plasmon resonance (Biacore), stopped flow spectroscopic, Soret CD, and analytical ultracentrifugation methodologies will be used to examine the rates and thermodynamics of the binding of various expressed CYP4A subfamily members with their common redox partner, NADPH-cytochrome P450 reductase, in the presence and absence cytochrome b5. In addition, mutations of putative surface residues on the CYP4A sub-family members will attempt to map the binding sites for these proteins. 3) Electron Transfer/Acceptor Properties of CYP4A Monooxygenase System: The rates of electron flux NADPH-cytochrome P450 reductase and CYP4As will be measured by stopped-flow spectrophotometry at wavelengths specific for the flavins or heme prosthetic groups to determine the effects on electron transfer of cyt b5, using various substrates, specific to each CYP4A.