Structure of bovine pancreatic cholesterol esterase at 1.6 Å:: Novel structural features involved in lipase activation
Structure of bovine pancreatic cholesterol esterase at 1.6 Å:: Novel structural features involved in lipase activation
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DOI：
10.1021/bi972989g
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发表时间：
1998-04-14
期刊：
影响因子：
2.9
通讯作者：
Stroud, RM
中科院分区：
文献类型：
--
作者：
Chen, JCH;Miercke, LJW;Stroud, RM
胰胆固醇酯酶是一种水解多种膳食脂质、介导胆固醇酯吸收并依赖于胆汁盐以获得最佳活性的酶，其结构被确定为1.6埃分辨率。在HEK 293细胞中表达突变以消除两个N-连接的糖基化位点（N187 Q/N361 Q）的全长构建体。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。突变酶是单体的，并表现出改善的均匀性，这有助于良好衍射晶体的生长。突变酶的晶体在空间群C2中生长，具有以下细胞尺寸：a = 100.42埃，B = 54.25埃，c = 106.34埃，β = 104.12度，在不对称单元中具有单体。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α-β水解酶折叠，在催化三联体周围具有不寻常的活性位点环境。蛋白质的疏水性C末端位于活性位点，使氧阴离子空穴远离生产性结合位点和催化性Ser 194。在其他甘油三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著特征。这两个结构特征，沿着胆盐依赖性的酶活性，暗示了一种新的脂肪酶激活模式。
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.