X-ray structure of human acid-β-glucosidase covalently bound to conduritol-B-epoxide -: Implications for Gaucher disease.
X-ray structure of human acid-β-glucosidase covalently bound to conduritol-B-epoxide -: Implications for Gaucher disease.
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DOI：
10.1074/jbc.m502799200
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发表时间：
2005-06-24
影响因子：
4.8
通讯作者：
Sussman, JL
中科院分区：
文献类型：
--
作者：
Premkumar, L;Sawkar, AR;Sussman, JL
戈谢病是一种遗传性代谢性疾病，由溶酶体酶酸性-β-葡萄糖苷酶 (GlcCerase) 突变引起。我们最近以 2.0 埃分辨率确定了 GlcCerase 的 X 射线结构（Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. (2003) EMBO Rep. 4, 704-709），现在已经解析了与一种不可逆抑制剂，conduritol-B-环氧化物（CBE）。晶体结构表明，CBE 与活性位点的结合不会引起 GlcCerase 的整体构象变化，并证实 Glu(340) 是催化亲核试剂。然而，在 GlcCerase-CBE 结构中仅观察到位于天然 GlcCerase 活性位点入口处的一对柔性环（残基 345-349 和 394-399）的两种可选构象之一，在这种构象中，CBE 可以接近活性位点。对这两种替代构象的动力学分析表明，这两个环充当活性位点入口处的盖子。环 394-399 中的一组突变支持了这种可能性，这些突变通过降低催化活性而导致戈谢病。此外，计算机突变分析表明，所有这些突变都稳定了限制进入活性位点的构象，从而为该环中的突变如何导致戈谢病提供了机制解释。
Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-beta-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 angstrom resolution (Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. ( 2003) EMBO Rep. 4, 704-709) and have now solved the structure of GlcCerase conjugated with an irreversible inhibitor, conduritol-B- epoxide ( CBE). The crystal structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu(340) is the catalytic nucleophile. However, only one of two alternative conformations of a pair of flexible loops ( residues 345-349 and 394-399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394-399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.