Protease inhibitors are crucial for the control of proteolytic activity in different physiological processes. However, some inhibitors do not show canonical enzyme recognition of the enzyme under certain conditions. In this work, we present evidence that indicates the formation of an active complex between the protease bovine α-chymotrypsin and the Tepary bean protease inhibitor (TBPI). The composition of the active chymotrypsin-TBPI complex (AC) was confirmed by three different methods: size-exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry. The kinetic parameters for the AC were similar to those of the enzyme alone, indicating that TBPI binding does not produce any large changes in chymotrypsin. The molecular model proposed here postulates that TBPI binds outside the active cleft of the protease, but near enough to hinder the binding of high molecular weight substrates into the active site. This model was experimentally supported by the inhibitory effect on casein as a substrate, and the unaltered protease activity when a small synthetic substrate was used. We also found that the formation of this complex provided the enzyme with extra stability in denaturing conditions or in the presence of a reducing agent. The chymotrypsin-TBPI complex exhibited higher stability, indicating that autolysis can be partially prevented. When the enzyme was first inactivated followed by the addition of the inhibitor, the activity of the protease was restored. We described a possible mechanism where a plant protease inhibitor binds outside the active site of the enzyme while increasing its stability.

中文翻译：

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三元豆蛋白酶抑制剂和α胰凝乳蛋白酶之间形成的非规范复合物的性质。

蛋白酶抑制剂对于控制不同生理过程中的蛋白水解活性至关重要。但是，某些抑制剂在某些条件下并未显示出对酶的规范酶识别。在这项工作中，我们提供的证据表明蛋白酶牛α-胰凝乳蛋白酶和三倍体豆蛋白酶抑制剂（TBPI）之间形成了活性复合物。活性胰凝乳蛋白酶-TBPI复合物（AC）的组成通过三种不同的方法确定：尺寸排阻色谱，聚丙烯酰胺凝胶电泳（PAGE）和质谱。AC的动力学参数与单独的酶相似，表明TBPI结合不会在胰凝乳蛋白酶上产生任何大的变化。这里提出的分子模型假定TBPI结合在蛋白酶的活性裂隙之外，但距离足够近，足以阻止高分子量底物结合到活性位点上。通过使用酪蛋白作为底物的抑制作用以及使用小的合成底物时蛋白酶的活性未改变，该模型在实验上得到了支持。我们还发现，该复合物的形成在变性条件下或在还原剂存在下为酶提供了额外的稳定性。胰凝乳蛋白酶-TBPI复合物具有更高的稳定性，表明可以部分防止自溶。当首先使酶失活，然后加入抑制剂时，蛋白酶的活性得以恢复。我们描述了一种可能的机制，其中植物蛋白酶抑制剂在增加酶的稳定性的同时与酶的活性位点结合。