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Structural analysis on the ligand specificity of γ-glutamylcysteine synthetase

γ-谷氨酰半胱氨酸合成酶配体特异性的结构分析

基本信息

批准号：
15580095
负责人：
ODA Jun'ichi
金额：
$ 2.11万
依托单位：
Fukui Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

γ-Glutamylcysteine synthetase (γGCS), a rate-limiting enzyme in glutathione biosynthesis, plays a central role in glutathione homeostasis and is a target for development of potential therapeutic agents against parasites and cancer.We have determined the crystal structures of E. coli γGCS complexed with a sulfoximine-based transition-state analog inhibitor and a classical inhibitor, buthionine sulfoximine (BSO), at resolutions of 2.1 and 2.3 A, respectively. The cysteine-binding site was identified; it is formed inductively at the transition state. In the unliganded structure, an openspace exists around the representative cysteine-binding site and is probably responsible for the competitive binding of glutathione. Upon binding of the transition-state analog, the side chains of Tyr-241 and Tyr-300 turn, forming a hydrogen-bonding triad with the carboxyl group of the inhibitors cysteine moiety, allowing this moiety to fit tightly into the cysteine-binding site with concomitant accommodation of its side chain into a shallow pocket. This movement is caused by a conformational change of a switch loop (residues 240-249). The side chain of Tyr-300 gives no electron density peak in the complex with BSO that has no carboxyl group essential to the formation of the hydrogen-bonding triad.Based on this crystal structure, the cysteine-binding sites of mammalian and parasitic γGCSs were predicted by multiple sequence alignment, although no significant sequence identity exists between the E. coli γGCS and its eukaryotic homologues. The identification of this cysteine-binding site provides important information for the rational design of novel γGCS inhibitors.

γ-谷氨酰半胱氨酸合成酶（γ-Glutamylcysteine synthetase，γ GCS）是谷胱甘肽生物合成的限速酶，在谷胱甘肽的体内平衡中起着重要作用，是开发抗寄生虫和抗肿瘤药物的靶点。coli γ GCS分别与磺酰亚胺类过渡态抑制剂和经典抑制剂丁硫基磺酰亚胺（BSO）复合，分离度分别为2.1和2.3 A。确定了半胱氨酸结合位点;它在过渡态诱导形成。在unliganded结构中，一个开放空间存在于代表性的半胱氨酸结合位点周围，可能是负责谷胱甘肽的竞争性结合。在结合过渡态类似物时，Tyr-241和Tyr-300的侧链转向，与抑制剂半胱氨酸部分的羧基形成氢键三单元组，使该部分紧密地配合到半胱氨酸结合位点中，同时将其侧链容纳到浅口袋中。这种移动是由开关环（残基240 - 249）的构象变化引起的。基于此晶体结构，通过多序列比对预测了哺乳动物和寄生虫γ GCSs的半胱氨酸结合位点，尽管E. coli γ GCS及其真核同源物。该半胱氨酸结合位点的鉴定为合理设计新型γ GCS抑制剂提供了重要信息。