The importance of being knotted: effects of the C-terminal knot structure on enzymatic and mechanical properties of bovine carbonic anhydrase II
The importance of being knotted: effects of the C-terminal knot structure on enzymatic and mechanical properties of bovine carbonic anhydrase II
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DOI：
10.1016/s0014-5793(02)02693-5
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发表时间：
2002-05-22
期刊：
影响因子：
3.5
通讯作者：
Ikai, A
中科院分区：
文献类型：
--
作者：
Alam, MT;Yamada, T;Ikai, A
为了更好地理解打结折叠模式对碳酸酐酶的酶和机械性质的贡献，我们用Cys替换了牛碳酸酐酶II的Gln-253，这使我们能够通过避免结收紧来测量蛋白质对抗拉伸变形的机械强度。令我们惊讶的是，表达的蛋白质原来含有两种构象异构体，一种能够结合酶抑制剂，另一种不能，这导致它们通过亲和色谱分离。在近紫外和远紫外圆二色谱和荧光光谱中，分离的构象彼此非常相似，并与野生型酶，表明它们都具有天然样构象。我们描述了新的证据，支持的概念，这两个构象之间的差异可能是有关的C-末端结形成的完整性。(C)2002年欧洲生物化学学会联合会。由Elsevier Science B. V.出版，版权所有。
In order to better understand the contribution of the knotted folding pattern to the enzymatic and mechanical properties of carbonic anhydrases, we replaced Gln-253 of bovine carbonic anhydrase II with Cys, which allowed us to measure the mechanical strength of the protein against tensile deformation by avoiding knot tightening. The expressed protein, to our surprise, turned out to contain two conformational isomers, one capable of binding an enzymatic inhibitor and the other not, which led to their separation through affinity chromatography. In near- and far-UV circular dichroism and fluorescence spectra, the separated conformers were very similar to each other and to the wild-type enzyme, indicating that they both had native-like conformations. We describe new evidence which supports the notion that the difference between the two conformers is likely to be related to the completeness of the C-terminal knot formation. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.