SQUALENE EPOXIDASE AND OXIDOSQUALENE CYCLASE FROM LIVER

来自肝脏的角鲨烯环氧化酶和氧化角鲨烯环化酶

• 批准号: 6014456
• 负责人: GLENN DOWNES PRESTWICH
• 金额: $ 5.64万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6014456
• 关键词: active sites; affinity labeling; animal genetic material tag; clone cells; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate complex; glycosylation; laboratory rabbit; laboratory rat; lanosterol; liver; mass spectrometry; matrix assisted laser desorption ionization; molecular cloning; nuclear magnetic resonance spectroscopy; oxygenases; polymerase chain reaction; protein purification; protein sequence; site directed mutagenesis; squalene; steroid metabolism; transferase

Squalene epoxidase (SE) and oxidosqualene cyclase (OSC) are the two key enzymes required for the production of lanosterol from an acyclic polyene precursor. Recently, these two enzymes, as well as squalene synthetase, have emerged as important new pharmaceutical targets for a dozen companies worldwide. We propose to continue our efforts to determine the molecular interactions involved in substrate binding and catalysis by exploiting the techniques of synthetic chemistry, protein biochemistry, molecular biology, and structural biology. Efficient affinity probes allowed purification and active site labeling of these two enzymes from rat and pig liver. SE from both sources has been affinity purified using a substrate mimic and photoaffinity labeled using a tritiated inhibitor analog: active site sequence analysis and physicochemical characterization have provided several unexpected results. OSC from both sources has been purified, modified with a tritium-labeled, mechanism-based inhibitor, and the active site amino acids sequenced. A novel aromatic-rich repeating motif and an Asp-rich cation binding region were identified. Using amino acids obtained from partially-sequenced peptide fragments, cloning of a cDNA for rat OSC will be attempted using both antibody screening and PCR amplification from a rat liver cDNA library in lambdaZAP. Use of rat cDNAs to complement the OSC-deficient erg7 yeast strain will also be attempted. The rat cDNA will be used to obtain a pig cDNA as well. Second, a similar strategy will be used for rat and pig SE when active site amino acid sequences are obtained. Third, OSC and SE cDNAs will be truncated to allow secretion of soluble forms, if possible, and expression systems will be developed to obtain multimilligram quantities for structural studies. Fourth, expression cassette PCR and site-directed mutagenesis will be employed to determine domains and residues respectively involved in substrate binding and catalysis. Fifth, catalytically-active domains of appropriate size will be crystallized and/or studied in liganded and unliganded states by multidimensional NMR. Sixth, OSC is extensively O-glycosylated. Studies of the native glycoproteins and derived glycopeptides will be undertaken using MALDI-MS- MS, and mutations will be made to determine importance of glycosylation sites. Seventh, effects of pressure on the rate and product distributions of OSC will be undertaken with recombinant enzymes.

沙列烯环氧化酶（SE）和氧化喹啉环酶（OSC）是两个键 从无环多烯生产型鼠溶所需要的酶 前体。最近，这两种酶以及小矛烯合成酶， 已经成为十二家公司的重要新药物目标 全世界。我们建议继续我们的努力来确定分子 通过利用与底物结合和催化有关的相互作用 合成化学，蛋白质生物化学，分子的技术 生物学和结构生物学。 有效的亲和力探针允许纯化和主动位点标记 这两种来自大鼠和猪肝脏的酶。来自两个来源的SE已经 使用底物纯化的亲和力模拟于使用 一个tri的抑制剂类似物：主动部位序列分析和 物理化学表征提供了几个意外的结果。 来自两个来源的OSC已被纯化，用tritium标记的修饰， 基于机理的抑制剂和活性位点氨基酸测序。一个 新型芳香富集的重复基序和富含ASP的阳离子结合区域 被确定。 使用从部分序列肽片段获得的氨基酸， 将尝试使用两种抗体来尝试将大鼠OSC的克隆 从大鼠肝cDNA文库中筛选和PCR扩增 Lambdazap。使用大鼠cDNA与OSC缺乏的ERG7酵母相辅相成 还将尝试应变。大鼠cDNA将用于获得猪 cDNA。其次，将用于大鼠和猪SE的类似策略 当获得活性位点氨基酸序列时。第三，OSC和SE 如果可能 将开发表达系统以获得多毫米 结构研究的数量。第四，表达盒式PCR和 将采用定向诱变来确定域和 残基分别涉及底物结合和催化。第五， 适当尺寸的催化活性域将结晶 和/或通过多维NMR在配体和非配体状态下进行研究。 第六，OSC被广泛的O-糖基化。本地的研究 将使用MALDI-MS-进行糖蛋白和衍生的糖肽 MS和将进行突变以确定糖基化的重要性 站点。第七，压力对速率和产品分布的影响 OSC将使用重组酶进行。