Prostaglandin 19-& 20-Hydroxylation by Cytochrome P450

前列腺素19-

• 批准号: 6543584
• 负责人: BETTIE SUE SILER MASTERS
• 金额: $ 30.28万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6543584
• 关键词: Escherichia coli; NADPH cytochrome c2 reductase; analytical ultracentrifugation; antibody; binding sites; chemical kinetics; complementary DNA; cytochrome P450; eicosanoid metabolism; electron transport; enzyme mechanism; enzyme substrate complex; heme; hydroxylation; isozymes; laboratory rabbit; molecular assembly /self assembly; mutant; oxygenases; prostaglandins; protein isoforms; protein protein interaction; site directed mutagenesis; stop flow technique; surface plasmon resonance; ultraviolet spectrometry

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.

描述（由申请方提供）：本研究计划将重点测定兔细胞色素P450的CYP 4A基因亚家族的4个成员的分子和动力学特性，这些成员主要或仅在ω-位羟基化中链和/或长链脂肪酸和类二十烷。细胞色素P4504 A4、4A 5、4A 6和4A 7在氨基酸水平上共享85%的序列同一性，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，但大量文献记载了它们通过控制血管张力在血流动力学调节中的作用。 例如，脑和肾微血管被浓度<10-10 M的ω-羟基花生四烯酸收缩。在前一个资助期内，这些酶被克隆并在E.大肠杆菌中使用每个同种型独特的技术。现在可以准备足够的数量用于实验，以了解它们的功能。这些密切相关的家族成员的产生允许它们之间的比较，就可以解释其底物偏好的氨基酸差异。此外，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ω-羟基化活性的显着活化。为了理解这些相互作用，提出了以下具体目的：1）CYP 4A与底物的相互作用：使用上述表达系统，将构建各种CYP 4A的其他突变，以比较独特的、同源的和在某些情况下相同的残基对所得蛋白质底物特异性活性的作用。在不存在和存在NADPH细胞色素P450还原酶和/或细胞色素b5的情况下，将通过停流分光光度法检查各种底物与CYP 4A的结合常数和结合速率。2)CYP 4A与其他蛋白质的相互作用：在存在和不存在细胞色素b5的情况下，将使用表面等离子体共振（Biacore）、停流光谱、Soret CD和分析性超离心方法来检查各种表达的CYP 4A亚家族成员与其共同氧化还原伴侣NADPH-细胞色素P450还原酶结合的速率和热力学。此外，CYP 4A亚家族成员上推定的表面残基的突变将尝试绘制这些蛋白质的结合位点。3)CYP 4A单加氧酶系统的电子转移/受体特性：将通过停流分光光度法在黄素或血红素辅基的特异性波长下测量NADPH-细胞色素P450还原酶和CYP 4A的电子通量速率，以确定使用各种底物对每种CYP 4A特异性对cyt b5的电子转移的影响。