Insights into the serine protease mechanism based on structural observations of the conversion of a peptidyl serine protease inhibitor to a substrate
Insights into the serine protease mechanism based on structural observations of the conversion of a peptidyl serine protease inhibitor to a substrate
复制标题
基于肽基丝氨酸蛋白酶抑制剂转化为底物的结构观察，深入了解丝氨酸蛋白酶机制
DOI：
10.1016/j.bbagen.2015.12.009
复制
发表时间：
2016
影响因子：
3
通讯作者：
Huang Mingdong
中科院分区：
文献类型：
--
作者：
Jiang Longguang;Andersen Lisbeth Moreau;Andreasen Peter A.;Chen Liqing;Huang Mingdong
丝氨酸蛋白酶是目前研究最多的一类酶。尽管进行了广泛的机理研究，但一些关键细节仍存在争议，例如，裂解产物如何在催化反应中释放。发现丝氨酸蛋白酶尿激酶型纤溶酶原激活物（uPA）的环肽基抑制剂（CSWRGLENHRMC, upain1）在替换单个残基时成为缓慢的底物并缓慢地裂解（W3A）。方法利用uPA肽的独特性质，用x射线晶体学方法报道了uPA与upin -1- w3a肽复合物在4种不同pH值下的高分辨率结构。结果在低pH （pH 4.6和5.5）条件下获得的结构中，环状肽upain-1-W3A保持完整，并保留在uPA的活性位点。在7.4时，肽的可剪切键被发现断裂，表明肽成为uPA底物。在pH为9.0时，底物的c端部分不再可见，只有占据S1口袋的P1残基被识别出来。结论upain-1-W3A是一种慢底物。此外，我们在酶活性位点的可剪键两侧清楚地鉴定出肽的裂解片段，显示出裂解肽的缓慢释放。这项工作表明，在第一步水解后，裂解的P '片段可能并不总是需要在第二步水解后快速释放。
BackgroundSerine proteases are one of the most studied group of enzymes. Despite the extensive mechanistic studies, some crucial details remain controversial, for example, how the cleaved product is released in the catalysis reaction. A cyclic peptidyl inhibitor (CSWRGLENHRMC, upain-1) of a serine protease, urokinase-type plasminogen activator (uPA), was found to become a slow substrate and cleaved slowly upon the replacement of single residue (W3A).MethodsBy taking advantage of the unique property of this peptide, we report the high-resolution structures of uPA in complex with upain-1-W3A peptide at four different pH values by X-ray crystallography.ResultsIn the structures obtained at low pH (pH 4.6 and 5.5), the cyclic peptide upain-1-W3A was found to be intact and remained in the active site of uPA. At 7.4, the scissile bond of the peptide was found cleaved, showing that the peptide became a uPA substrate. At pH 9.0, the C-terminal part of the substrate was no longer visible, and only the P1 residue occupying the S1 pocket was identified.ConclusionsThe analysis of these structures provides explanations why the upain-1-W3A is a slow substrate. In addition, we clearly identified the cleaved fragments of the peptide at both sides of the scissile bond in the active site of the enzyme, showing a slow release of the cleaved peptide.General significanceThis work indicates that the quick release of the cleaved P′ fragment after the first step of hydrolysis may not always be needed for the second hydrolysis.