The catalytic site residues and interfacial binding of human pancreatic lipase.
The catalytic site residues and interfacial binding of human pancreatic lipase.
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DOI：
10.1016/s0021-9258(18)41893-5
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发表时间：
1992-08
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影响因子：
--
通讯作者：
Mark E Lowe
中科院分区：
文献类型：
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作者：
Mark E Lowe
在这项研究中，必需的丝氨酸残基和其他2个氨基酸在人胰甘油三酯脂肪酶（三酰甘油酰基水解酶，EC 3.1.1.3）的酶的催化位点或界面结合位点的贡献进行了测试。通过对人胰脂肪酶的cDNA进行位点特异性诱变，在Ser 153、His 264和Asp 177处进行氨基酸取代。突变体cDNA在转染的COS-1细胞中表达。通过蛋白质印迹分析检查培养基和细胞中是否存在胰脂肪酶。测定了表达蛋白对三油酸甘油酯的活性和界面结合。Ser 153突变的蛋白质由细胞分泌并与界面结合，但没有可检测的活性。将His 264改变为亮氨酸或将Asp 177改变为天冬酰胺也产生无活性的脂肪酶。用谷氨酸取代Asp 177产生了一种活性蛋白。这些结果表明，Ser 153参与的胰脂肪酶的催化位点，并不是至关重要的界面结合。此外，His 264和Asp 177在催化中的重要作用被证明。人胰脂肪酶中存在一个类似于丝氨酸蛋白酶的Ser-His-Asp催化三联体。
In this study, the essential serine residue and 2 other amino acids in human pancreatic triglyceride lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) were tested for their contribution to the enzyme's catalytic site or interfacial binding site. By site-specific mutagenesis of the cDNA for human pancreatic lipase, amino acid substitutions were made at Ser153, His264, and Asp177. The mutant cDNAs were expressed in transfected COS-1 cells. Both the medium and the cells were examined for the presence of pancreatic lipase by Western blot analysis. The activity of the expressed proteins against triolein and the interfacial binding was measured. Proteins with mutations in Ser153 were secreted by the cells and bound to interfaces but had no detectable activity. Changing His264 to a leucine or Asp177 to an asparagine also produced inactive lipase. Substituting glutamic acid for Asp177 produced an active protein. These results demonstrate that Ser153 is involved in the catalytic site of pancreatic lipase and is not crucial for interfacial binding. Moreover, the essential roles of His264 and Asp177 in catalysis were demonstrated. A Ser-His-Asp catalytic triad similar to that present in serine proteases is present in human pancreatic lipase.