SQUALENE EPOXIDASE AND OXIDOSQUALENE CYCLASE FROM LIVER

角鲨烯环氧酶（SE）和氧化角鲨烯环化酶（OSC）是两个关键酶， 由无环多烯生产羊毛甾醇所需的酶 先驱最近，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 国际吧我们建议继续努力确定 底物结合和催化的相互作用， 合成化学、蛋白质生物化学、分子生物学技术 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肝。这两个来源的SE一直是 使用底物模拟物进行亲和纯化，并使用 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几个意想不到的结果。 来自两种来源的OSC已经被纯化，用氚标记的， 机制为基础的抑制剂，和活性位点的氨基酸测序。一 新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 被确认了身份 使用从部分测序的肽片段获得的氨基酸， 将使用两种抗体尝试克隆大鼠OSC的cDNA 从大鼠肝脏cDNA文库中筛选和PCR扩增， 快去ZAP大鼠cDNA用于补充OSC缺陷型erg 7酵母的用途 也将尝试应变。大鼠cDNA将用于获得猪 cDNA也是。第二，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。第三，OSC和SE 将cDNA截短以允许可溶形式的分泌，如果可能， 并将开发表达系统以获得多毫克 结构研究的数量。第四，表达盒PCR和 将采用定点诱变来确定结构域， 分别参与底物结合和催化的残基。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研究。 第六，OSC是广泛O-糖基化的。对原住民的研究 糖蛋白和衍生的糖肽将使用MALDI-MS进行， MS，并进行突变以确定糖基化的重要性 网站.第七，压力对速率和产物分布的影响 将用重组酶进行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.