The structure of an allosamidin complex with the Coccidioides immitis chitinase defines a role for a second acid residue in substrate-assisted mechanism
The structure of an allosamidin complex with the Coccidioides immitis chitinase defines a role for a second acid residue in substrate-assisted mechanism
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DOI：
10.1016/s0022-2836(02)00444-8
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发表时间：
2002-07-05
影响因子：
5.6
通讯作者：
Robertus, JD
中科院分区：
文献类型：
--
作者：
Bortone, K;Monzingo, AF;Robertus, JD
阿勒萨米丁是已知的18类几丁质酶的抑制剂。我们发现，别沙米丁是球孢子虫真菌几丁质酶CiX1的竞争性抑制剂，其K-I为60 nM。我们报道了该络合物的X射线结构，结果表明，当抑制剂结合时，Asp169的侧链发生旋转，与恶唑阳离子形成离子对。作用机制被认为包括Glu171质子化离开基团和底物被糖乙酰氨基部分辅助形成恶唑啉类中间体。我们将这两个氨基酸残基转化为相应的酰胺，发现每个突变都有效地取消了酶的活性。X射线结构表明，突变酶保持了基本的野生型结构，突变活性的丧失是由于它们的化学性质改变。别三胺的高亲和力，以及它与假定的反应中间体的相似性，表明它是一种过渡态类似物。这有助于验证我们的论点，即Asp169的作用是静电稳定反应过渡态。(C)2002爱思唯尔科学有限公司。保留所有权利。
Allosamidin is a known inhibitor of class 18 chitinases. We show that allosamidin is a competitive inhibitor of the fungal chitinase CiX1 from Coccidioides immitis, with a K-i of 60 nM. We report the X-ray structure of the complex and show that upon inhibitor binding the side-chain of Asp169 rotates to form an ion pair with the oxazolinium cation. The mechanism of action is thought to involve protonation of the leaving group by Glu171 and substrate assistance by the sugar acetamido moiety to form an oxazoline-like intermediate. We converted both amino acid residues to the corresponding amide and found that each mutation effectively abolishes enzyme activity. X-ray structures show the mutant enzymes retain the basic wild-type structure and that the loss of mutant activity is due to their altered chemical properties. The high affinity of allosamidin, and its similarity to the putative reaction intermediate, suggests it is a transition state analog. This helps validate our contention that the role of Asp169 is to electrostatically stabilize the reaction transition state. (C) 2002 Elsevier Science Ltd. All rights reserved.