Departamento de Biotecnología y Bioquímica del Cinvestav Unidad Irapuato, Km. 9.6 Libramiento Norte Carretera Irapuato-León, 36824, León, Mexico.
Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Av. de las Ciencias S/N, 76230, Juriquilla, Querétaro, Mexico.
Protein J. 2019 Aug;38(4):435-446. doi: 10.1007/s10930-019-09863-2.
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.
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。然而，在某些条件下，一些抑制剂并不表现出对酶的典型酶识别。在这项工作中，我们提供了证据表明蛋白酶牛α-糜蛋白酶和豇豆蛋白酶抑制剂（TBPI）之间形成了一个活性复合物。通过三种不同的方法证实了活性糜蛋白酶-TBPI 复合物（AC）的组成：分子筛层析、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 结合不会使糜蛋白酶发生任何大的变化。这里提出的分子模型假设 TBPI 结合在蛋白酶的活性裂缝之外，但足够接近以阻止高分子量底物结合到活性位点。该模型通过对酪蛋白作为底物的抑制作用以及当使用小的合成底物时蛋白酶活性不变得到了实验支持。我们还发现，这种复合物的形成赋予了酶在变性条件下或存在还原剂时额外的稳定性。糜蛋白酶-TBPI 复合物表现出更高的稳定性，表明自溶可以部分得到预防。当酶首先失活然后加入抑制剂时，蛋白酶的活性得到恢复。我们描述了一种可能的机制，其中植物蛋白酶抑制剂结合在酶的活性位点之外，同时增加其稳定性。
