SQUALENE EPOXIDASE AND OXIDOSQUALENE CYCLASE FROM LIVER

角鲨烯环氧酶(SE)和氧化角鲨烯环化酶(OSC)是其中的两个关键酶 从无环多烯生产羊毛甾醇所需的酶 前身。最近，这两种酶以及角鲨烯合成酶， 已经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 参与底物结合和催化的相互作用 合成化学、蛋白质生物化学、分子技术 生物学和结构生物学。 高效亲和探针的纯化和活性部位标记 这两种酶来自大鼠和猪的肝脏。来自两个来源的Se一直是 用底物模拟纯化的亲和力和用 一种氚抑制剂类似物：活性位点序列分析和 物理化学表征提供了几个意想不到的结果。 来自这两个来源的OSC已经被提纯，用氚标记的， 基于机理的抑制剂，并对活性部位的氨基酸进行了测序。一个 新的富芳重复基序和富含天冬氨酸的阳离子结合区 都被确认了。 使用从部分测序的多肽片段中获得的氨基酸， 我们将尝试用这两种抗体克隆大鼠口腔鳞状细胞癌的基因 大鼠肝脏cDNA文库的筛选及PCR扩增 LambdaZAP。利用大鼠的cDNA来补充OSC缺陷的erg7酵母 还将尝试应变。大鼠的cDNA将被用来获得一头猪 C DNA也是如此。其次，类似的策略将用于老鼠和猪的SE 当获得活性部位的氨基酸序列时。第三，OSC和SE 如果可能的话，cDNA将被截断，以允许可溶性形式的分泌， 和表达系统将被开发以获得多微米图 用于结构研究的数量。第四，表达盒聚合酶链式反应和 将使用定点突变来确定结构域和 残基分别参与底物结合和催化。第五, 适当大小的催化活性结构域将被结晶 和/或用多维核磁共振研究配位和非配位状态。 第六，OSC是广泛的O-糖基化的。原住民研究 糖蛋白和衍生的糖肽将使用MALDI-MS进行研究。 MS，并进行突变以确定糖基化的重要性 网站。第七，压力对速率和产品分布的影响 将使用重组酶进行OSC的研究。

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.