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首页> 外文期刊>Biochemistry >Structure of bovine pancreatic cholesterol esterase at 1.6 angstrom: Novel structural features involved in lipase activation
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Structure of bovine pancreatic cholesterol esterase at 1.6 angstrom: Novel structural features involved in lipase activation

机译：牛胰腺胆固醇酯酶在1.6埃的结构：涉及脂肪酶激活的新结构特征

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摘要

The structure of pancreatic cholesterol esterase, an enzyme that hydrolyzes a wide variety of dietary lipids, mediates the absorption of cholesterol esters, and is dependent on bile salts for optimal activity, is determined to 1.6 Angstrom resolution. A full-length construct, mutated to eliminate two N-linked glycosylation sites (N187Q/N361Q), was expressed in HEK 293 cells. Enzymatic activity assays show that the purified, recombinant, mutant enzyme has activity identical to that of the native, glycosylated enzyme purified from bovine pancreas. The mutant enzyme is monomeric and exhibits improved homogeneity which aided in the growth of well-diffracting crystals. Crystals of the mutant enzyme grew in space group C2, with the following cell dimensions: a = 100.42 Angstrom, b = 54.25 Angstrom, c = 106.34 Angstrom, and beta = 104.12 degrees, with a monomer in the asymmetric unit. The high-resolution crystal structure of bovine pancreatic cholesterol esterase (R-cryst = 21.1%; R-free = 25.0% to 1.6 Angstrom resolution) shows an alpha-beta hydrolase fold with an unusual active site environment around the catalytic triad. The hydrophobic C terminus of the protein is lodged in the active site, diverting the oxyanion hole away from the productive binding site and the catalytic Ser194. The amphipathic, helical lid found in other triglyceride lipases is truncated in the structure of cholesterol esterase and therefore is not a salient feature of activation of this lipase. These two structural features, along with the bile salt-dependent activity of the enzyme, implicate a new mode of lipase activation. [References: 43]
机译：胰胆甾醇酯酶的结构被确定为1.6埃分辨率，该酶可水解多种饮食脂质，介导胆甾醇酯的吸收，并依赖胆汁盐获得最佳活性。在HEK 293细胞中表达了全长构建体，该构建体经突变消除了两个N-连接的糖基化位点（N187Q / N361Q）。酶活性测定表明，纯化的重组突变酶具有与从牛胰腺纯化的天然糖基化酶相同的活性。突变酶是单体酶，并显示出改善的均质性，这有助于良好衍射晶体的生长。突变酶的晶体在C2空间群中生长，其细胞尺寸如下：a = 100.42埃，b = 54.25埃，c = 106.34埃，和β= 104.12度，单体位于不对称单元中。牛胰腺胆固醇酯酶的高分辨率晶体结构（R-cryst = 21.1％; R-free = 25.0％至1.6埃分辨率）显示了α-β水解酶折叠，催化三联体周围有异常的活性位点环境。蛋白质的疏水性C末端位于活性位点，使氧阴离子孔远离生产性结合位点和催化性Ser194。在其他甘油三酸酯脂肪酶中发现的两亲性螺旋盖在胆固醇酯酶的结构中被截短，因此不是该脂肪酶活化的显着特征。这两个结构特征，以及该酶的胆汁盐依赖性活性，暗示了脂肪酶激活的新模式。 [参考：43]

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