EFFECT OF SERPIN BINDING ON ACTIVE SITE OF TARGET PROTEINASE

人β1-蛋白水解酶抑制剂与大鼠胰蛋白酶的结合 核磁共振波谱显示提高了pKa？在他的57岁的现役 但不破坏His57和Asp102之间的氢键。 Asp189对丝氨酸突变体也有类似的核磁共振结果 大鼠胰酶，比野生型胰酶更稳定，对 碱基残基突变所致的自身蛋白降解 专一性的口袋里。这个突变体被用于进一步的研究 目的是确定构象转变的程度 伴随着丝氨酸结合的胰酶，并导致破坏 蛋白水解酶的催化活性使抑制剂复合体 被困在酰基酶的中间阶段。的稳定性 大鼠胰酶对热变性的影响较低。 游离酶比含有β-1-蛋白水解酶抑制剂的复合体中的活性低。这 表明该复合体含有广泛的蛋白质-蛋白质 稳定整体折叠的相互作用。另一方面， 先前的研究表明，体内的蛋白酶 丝氨酸-蛋白酶复合体变得更容易受到限制 蛋白质分解，表明构象变化 伴随绑定导致暴露在 酵素。这种构象变化的存在 通过对速度的研究，证实了这里的复杂形成 通过添加二硫代硫醇裂解二硫键。本研究 据透露，尽管胰酶在体内的稳定性增加 复杂，它的一个或多个二硫键变得容易得多 减少了。我们认为与之形成络合物的过程 -1-蛋白酶抑制剂将胰酶D189S转化为无活性的、松散的 结构，它是酶的“构象陷阱”， 防止催化脱酰化。还有人提议，塑料 胰酶激活区(S)可能起着至关重要的作用 在这种抑制剂诱导的结构重排中。

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.