Active site binding loop stabilization in the subtilisin inhibitor eglin c: structural and functional studies on specifically designed mutants in complex with subtilisin and the uncomplexed inhibitor.
Active site binding loop stabilization in the subtilisin inhibitor eglin c: structural and functional studies on specifically designed mutants in complex with subtilisin and the uncomplexed inhibitor.
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枯草杆菌蛋白酶抑制剂eglin c中的活性位点结合环稳定性：对与枯草杆菌蛋白酶和未复合的抑制剂复合的专门设计的突变体进行结构和功能研究。
DOI：
10.1007/978-1-4613-0319-0_6
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发表时间：
1996
影响因子：
--
通讯作者：
M. Grütter
中科院分区：
文献类型：
--
作者：
K. Hipler;J. Priestle;J. Rahuel;M. Grütter
如从与蛋白酶复合的X射线晶体结构和从NMR研究中已知的，丝氨酸蛋白酶抑制剂eglin c具有楔形形状，其具有疏水核心和溶剂暴露的活性位点结合环，其通过非共价核心-结合环相互作用的网络稳定。先前的研究暗示了P1 '残基Asp-46对于eglin c对丝氨酸蛋白酶如枯草杆菌蛋白酶的结合环稳定性和高抑制效力的关键作用。在本研究中，通过用天冬酰胺、戊二酸和谷氨酰胺替换野生型Asp-46来调节特异性eglin核心-结合环相互作用的形成。这些突变体的X-射线晶体结构与枯草杆菌蛋白酶的复合物中解决，并对这种酶的抑制效力进行了测定。我们的研究结果意味着减少的抑制效力下降的核心结合环的相互作用。我们成功地结晶游离野生型eglin c。1.95埃的X-射线晶体结构表明，从游离形式到结合形式的eglin的过渡是伴随着一个协调的构象变化的结合环，这意味着一个诱导适合可访问的酶表面。除了结合环结构域和eglin表面上的一些残基外，在未复合和结合形式的抑制剂之间观察到的差异很小。
As known from the x-ray crystal structure in complex with a proteinase and from NMR studies, the serine proteinase inhibitor eglin c has a wedge-like shape with a hydrophobic core and a solvent exposed active site binding loop which is stabilized by a network of non-covalent core-binding loop interactions. Previous studies implied a crucial role of the P1'-residue Asp-46 for binding loop stabilization and high inhibitory potency of eglin c towards serine proteinases such as subtilisin. In the present study, the formation of specific eglin core-binding loop interactions was modulated by replacing the wildtype Asp-46 by asparagine, glutarnate and glutamine. The x-ray crystal structures of these mutants were solved in complex with subtilisin, and the inhibitory potency towards this enzyme was determined. Our results imply a reduction of inhibitory potency with declining core-binding loop interactions. We succeeded in crystallizing free wildtype eglin c. The 1.95 angstroms x-ray crystal structure indicates that the transition from the free to the bound form of eglin is accompanied by a concerted conformational change in the binding loop, implying an induced fit to the accessible enzyme surface. Except for the binding loop domain and a few residues on the surface of eglin, the differences observed between the uncomplexed and bound form of the inhibitor are only small.
序列特异性 1H NMR 归属和eglin c 的二级结构。
影响因子：
2.9
作者：
Hyberts,SG;Wagner,G
通讯作者：
Wagner,G