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首页> 外文期刊>Biochemistry >Effects of serpin binding on the target proteinase: global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site.
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Effects of serpin binding on the target proteinase: global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site.

机译：丝氨酸蛋白酶抑制蛋白结合对靶蛋白酶的影响：整体稳定，局部增加结构的灵活性，并保留在活性位点的氢键。

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摘要

The binding of human alpha1-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 enzyme intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with alpha1-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 disulfide linkages by added dithiothreitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easily reduced. We suggest that the process of complex formation with alpha1-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.
机译：NMR光谱显示人α1-蛋白酶抑制剂与大鼠胰蛋白酶的结合可在活性位点上提高His57的pKa'，但不会破坏His57与Asp102之间的氢键。对于大鼠胰蛋白酶的Asp189到丝氨酸突变体，观察到了相似的NMR结果，由于特异性口袋底部的残基突变，该突变体比野生型胰蛋白酶对自身蛋白水解稳定得多。该突变体用于进一步研究，目的是确定胰蛋白酶中伴随丝氨酸蛋白酶抑制蛋白结合的构象转变的程度，并导致蛋白酶催化活性的破坏，从而使抑制剂复合物陷于酰基酶的中间阶段。发现在自由酶中大鼠胰蛋白酶对热变性的稳定性低于在具有α1-蛋白酶抑制剂的复合物中的稳定性。这表明该复合物包含稳定整体折叠的广泛的蛋白质-蛋白质相互作用。另一方面，先前的研究表明，丝氨酸蛋白酶抑制蛋白-蛋白酶复合物中的蛋白酶变得更容易受到有限的蛋白水解作用，这表明伴随结合的构象变化导致酶中易感环的暴露。通过研究添加的二硫苏糖醇对二硫键的裂解速率，已经证实了复合物形成时这种构象变化的存在。这项研究表明，尽管胰蛋白酶在复合物中的稳定性提高了，但其二硫键中的一个或多个桥变得更容易还原。我们建议与alpha1蛋白酶抑制剂形成复杂的过程将胰蛋白酶D189S转换为无活性的，松散的结构，这是防止催化脱酰作用的酶的“构象陷阱”。还提出胰蛋白酶的激活结构域的塑性区可能在该抑制剂诱导的结构重排中起关键作用。

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