Probing conformational plasticity of the activation domain of trypsin:: The role of glycine hinges
Probing conformational plasticity of the activation domain of trypsin:: The role of glycine hinges
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DOI：
10.1021/bi701454e
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发表时间：
2008-02-12
期刊：
影响因子：
2.9
通讯作者：
Graf, Laszlo
中科院分区：
文献类型：
--
作者：
Gombos, Linda;Kardos, Jozsef;Graf, Laszlo
胰蛋白酶样丝氨酸蛋白酶在止血、细胞凋亡、信号转导、生殖、免疫应答、基质重塑、发育和分化等多种生理过程中发挥重要作用。所有这些蛋白酶都有一个有趣的激活机制，包括酶原的未折叠结构域（激活结构域）向活性酶的折叠结构域的转变。在这种构象变化过程中，激活结构域片段围绕高度保守的甘氨酸铰链移动。在本研究中，通过位点定向诱变，铰链甘氨酸被丙氨酸残基取代。研究了这些突变对酶样构象和活性构象相互转化以及催化活性的影响。突变型胰蛋白酶表现出不同程度的酶原样结构，其特征是某些激活结构域片段的灵活性增加，n端更容易接近，底物结合位点变形。我们的研究结果表明，突变阻碍了胰蛋白酶原向胰蛋白酶的转化，导致无活性的酶原样构象和活性酶构象之间的平衡向无活性状态转移。然而，我们的数据也显示，各种突变体的非活性构象彼此不同。底物类似物的结合使构象平衡向活性酶转移，因为抑制形式的胰蛋白酶突变体表现出与野生型酶相似的结构特征。突变体的催化活性与活性位点的正确构象有关，这可以通过n端和自溶环的不同构象来支持。瞬态动力学测量证实了在底物结合之前发生的非活性到活性构象转变的存在。
Trypsin-like serine proteases play essential roles in diverse physiological processes such as hemostasis, apoptosis, signal transduction, reproduction, immune response, matrix remodeling, development, and differentiation. All of these proteases share an intriguing activation mechanism that involves the transition of an unfolded domain (activation domain) of the zymogen to a folded one in the active enzyme. During this conformational change, activation domain segments move around highly conserved glycine hinges. In the present study, hinge glycines were replaced by alanine residues via site directed mutagenesis. The effects of these mutations on the interconversion of the zymogen-like and active conformations as well as on catalytic activity were studied. Mutant trypsins showed zymogen-like structures to varying extents characterized by increased flexibility of some activation domain segments, a more accessible N-terminus and a deformed substrate binding site. Our results suggest that the trypsinogen to trypsin transition is hindered by the mutations, which results in a shift of the equilibrium between the inactive zymogen-like and active enzyme conformations toward the inactive state. Our data also showed, however, that the inactive conformations of the various mutants differ from each other. Binding of substrate analogues shifted the conformational equilibrium toward the active enzyme since inhibited forms of the trypsin mutants showed similar structural features as the wild-type enzyme. The catalytic activity of the mutants correlated with the proper conformation of the active site, which could be supported by varying conformations of the N-terminus and the autolysis loop. Transient kinetic measurements confirmed the existence of an inactive to active conformational transition occurring prior to substrate binding.