EFFECT OF SERPIN BINDING ON ACTIVE SITE OF TARGET PROTEINASE

人类的束缚？1-大鼠胰蛋白酶抑制剂 显示通过NMR光谱提高pKa？ His 57在活动中 但不破坏His 57和Asp 102之间的氢键。 对于Asp 189至丝氨酸突变体，观察到类似的NMR结果。 大鼠胰蛋白酶，其比野生型胰蛋白酶稳定得多， 由于碱基残基突变而引起的蛋白质自身水解 特异性口袋 该突变体用于进一步的研究 目的是确定构象转变的程度， 胰蛋白酶，伴随丝氨酸蛋白酶抑制剂结合，并导致破坏 蛋白酶的催化活性，使得抑制剂复合物 被困在酰基酶中间阶段。 的稳定性 大鼠胰蛋白酶对热变性被发现是较低的， 游离酶比在复杂的？1-蛋白酶抑制剂 这 表明该复合物含有大量的蛋白质-蛋白质 稳定整体折叠的相互作用。 另一方面，在一项研究中， 以前的研究表明， 蛇毒蛋白酶-蛋白酶复合物变得更容易受到有限的 蛋白质水解，这表明构象变化， 伴随着结合导致暴露的易感环在 酵素 这种构象变化的存在， 复合物的形成已被证实在这里通过调查的速度 通过添加二硫代糖醇裂解二硫键。 本研究 显示，尽管胰蛋白酶在细胞中的稳定性增加， 复杂，一个或多个二硫桥变得更加容易 降低 我们认为，复合物的形成过程 ? 1-蛋白酶抑制剂将胰蛋白酶D189 S转化为一种无活性的，松散的 结构，其作为酶的“构象陷阱”， 防止催化脱酰作用。 还建议塑料 胰蛋白酶激活结构域的区域可能起关键作用 在这个由通道引起的结构重排中。

The binding of human ?1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pKa? of His57 in the active site but not to disrupt the hydrogen bond between His57 and Asp102. Similar NMR results were observed for the Asp189 to serine mutant of rat trypsin, which is much more stable than wild-type trypsin against autoproteolysis as the result of mutation of the residue at the base of the specificity pocket. This mutant was used in further studies aimed at determining the extent of the conformational transition in trypsin that accompanies serpin binding and leads to disruption of the catalytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzymes intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with ?1-proteinase inhibitor. This suggests that the complex contains extensive protein-protein interactions that stabilize overall folding. On the other hand, previous investigations have shown that the proteinase in serpin-proteinase complexes becomes more susceptible to limited proteolysis, suggesting that the conformational change that accompanies binding leads to the exposure of susceptible loops in the enzyme. The existence of this type of conformational change upon complex formation has been confirmed here by investigation of the rate of cleavage of disulfie linkages by added dithiotheitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easity reduced. We suggest that the process of complex formation with ?1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a "conformational trap" of the enzyme that prevents catalytic deacylation. It is also proposed that plastic region(s) of the activation domain of trypsin may play a crucial role in this inhibitor-induced structural rearrangement.