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.

角鲨烯环氧化酶（SE）和氧化角鲨烯环化酶（OSC）是两个关键的酶 从无环多烯生产羊毛甾醇所需的酶 前驱。最近，这两种酶以及角鲨烯合成酶， 已成为十几家公司的重要新制药目标 全世界。我们建议继续努力确定分子 通过利用参与底物结合和催化的相互作用 合成化学技术、蛋白质生物化学、分子 生物学和结构生物学。 高效的亲和探针允许纯化和活性位点标记 这两种酶来自大鼠和猪的肝脏。来自两个来源的 SE 已 使用底物模拟物进行亲和纯化，并使用光亲和标记 氚化抑制剂类似物：活性位点序列分析和 物理化学表征提供了一些意想不到的结果。 两种来源的 OSC 均已纯化，并用氚标记的、 基于机制的抑制剂，并对活性位点氨基酸进行了测序。一个 新颖的富含芳香族的重复基序和富含天冬氨酸的阳离子结合区域 被识别出来。 使用从部分测序的肽片段获得的氨基酸， 将尝试使用两种抗体克隆大鼠 OSC 的 cDNA 大鼠肝脏 cDNA 文库的筛选和 PCR 扩增 lambdaZAP。使用大鼠 cDNA 来补充 OSC 缺陷的 erg7 酵母 也将尝试应变。大鼠 cDNA 将用于获得猪 cDNA也是如此。其次，类似的策略将用于大鼠和猪 SE 当获得活性位点氨基酸序列时。三、OSC和SE 如果可能的话，cDNA 将被截短以允许分泌可溶形式， 将开发表达系统以获得数毫克 结构研究的数量。四、表达盒PCR和 将采用定点诱变来确定结构域和 残基分别参与底物结合和催化。第五， 适当大小的催化活性域将被结晶 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 O-糖基化。本土研究 糖蛋白和衍生糖肽将使用 MALDI-MS-进行 MS，并将进行突变以确定糖基化的重要性 网站。七、压力对费率和产品分布的影响 OSC 将使用重组酶进行。