Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.
Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.
复制标题
扰动胰蛋白酶中 Asp102 的极地环境：替换保守 Ser214 的后果。
DOI：
10.1021/bi00127a005
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Fletterick,RJ
中科院分区：
文献类型：
--
作者：
McGrath,ME;Vásquez,JR;Craik,CS;Yang,AS;Honig,B;Fletterick,RJ
胰蛋白酶的催化能力很大程度上来自于Aspl02不寻常的隐藏的、带电的侧链。极性洞穴提供了维持埋藏电荷的稳定性，并且它的特征是与Aspl02相邻的保守氨基酸Ser214。大鼠阴离子胰蛋白酶中的Ser214已被Ala、Glu和Lys所取代，这些变化的后果已经确定。Glu和Lys变体胰蛋白酶的三维结构显示，新的214个侧链被隐藏。胰蛋白酶S214K的2.2-A晶体结构（R= 0.150）表明，lys214占据了Ser214和埋水分子在埋极洞中的位置。Lys214-Nf是溶剂不可接近的，与催化Aspl02的距离小于5 A。胰蛋白酶S214E中Glu214侧链（2.8 k， R=0.168）有两种构象。在主链中，S214E中的Glu羧酸盐与Asp 102形成氢键。等电聚焦分析结果表明，胰蛋白酶S214K的等电点与胰蛋白酶的等电点明显不同，对应于胰蛋白酶多带一个正电荷。动力学参数表明，与胰蛋白酶相比，S214K对三肽酰胺底物的催化活性为1%，而S214E的活性为44%。静电电位计算证实了Lys214上的电荷，并且与动力学结果一致，表明Lys214的存在干扰了Aspl02的静电电位。
Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Aspl02. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Aspl02. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R= 0.150) of trypsin S214K shows that Lys214occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-Nf is solvent inaccessible and is less than 5 A from the catalytic Aspl02. The side chain of Glu214 (2.8 k, R=0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp 102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter demonstrates that, compared to trypsin, S214K has 1% of the catalyticactivity on a tripeptide amide substrateand S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Aspl02.