Studies on three-dimensional structures of the glycolytic enzymes responsible for the lysosomal diseases

负责溶酶体疾病的糖酵解酶的三维结构研究

基本信息

批准号：
12470486
负责人：
SATOW Yoshinori
金额：
$ 9.98万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Fabry disease is an X-linked hereditary disorder of lysosomal α-galactosidase. This enzyme hydrolyzes terminal α galactoside linkages in various glycolipids, and its genetic mutations decrease enzymatic activities which result in deficiencies of glycolipid metabolism and hence accumulates such substrate as glycosphingolipid of globotriaosylceramide in heart and other organs. The molecular lesion of lysosomal α-N-acetylgalactosaminidase causes another form of angiokeratoma. In order to elucidate three-dimensional structural bases of these diseases, both the enzymes have been expressed and subjected to crystallographic studies.A recombinant of human α-galactosidase was first expressed in the yeast Pichia pastoris that successfully secretes a glycosilated and catalytically active enzyme into a culture broth. Three recombinants for the mutated enzymes were similarly obtained and characterized by biophysical and biochemical analyzes. Crystals of the enzymes both in free forms and in complex … More es with a inhibitor of 1-deoxygalactonojirimycin were obtained. X-ray diffraction data from these crystals were collected using laboratory and synchrotron sources, and analyzed with the molecular replacement method.The three-dimensional structures thus obtained show that α-galactosidase is homodimeric, consisting of a β-sheet domain and an α/β barrel domain in which the active site is located. The structures of the two recombinants responsible for the variant-form disease indicate that their differences from the structure of the normal enzyme are very small and localized only near the mutated amino acids, and their characterizations indicate that they attain nearly normal levels of the activities but lose their activities rapidly. The structure of the recombinant causing the classical-form disease show that the side chain of the mutated amino-acid blocks the active site and completely hinders the activity. A recombinant of human α-N-acetylgalactosaminidase was similarly expressed, purified, and crystallized. Optimization of qualities of the obtained crystals is in progress so as to elucidate the structure of this enzyme. Less

Fabry病是溶酶体α-半乳糖苷酶的X连锁遗传性疾病。这种酶在各种糖脂中水解了末端α-半乳糖苷链接，其遗传突变降低了酶促活性，从而导致糖脂代谢的缺乏，因此会在心脏和其他Organs中累积糖脂蛋白酶糖脂糖脂的糖磷脂果。溶酶体α-N-乙酰乳糖胺酶的分子病变会引起另一种形式的血管瘤。为了阐明这些疾病的三维结构碱基，这两种酶均已表达并进行了晶体学研究。人α-半乳糖苷酶的重组者首先在酵母菌pichia Pastoris中表达，成功地分泌了一种糖化和催化活跃的活跃的活跃的enzyzyme，并将其分泌到培养物中。类似地获得了突变酶的三种重组，并通过生物物理和生化分析进行了特征。酶的晶体都以游离形式和复杂性……具有1-脱氧甲状腺乳糖二霉素抑制剂的更多ES。使用实验室和同步源收集了从这些晶体中的X射线衍射数据，并用分子替换方法进行了分析。因此获得的三维结构表明，α-乳糖苷酶是同型二维结构，由β-片状域和由α/β桶域组成，由α/β桶域组成。负责变异疾病的两种重组者的结构表明，它们与正常酶结构的差异非常小，并且仅在突变的氨基酸附近局部化，并且它们的特征表明它们几乎达到了活动水平，但迅速失去了活动。引起经典疾病的重组的结构表明，突变的氨基酸的侧链阻止了活性位点并完全阻碍了活性。类似表达，纯化和结晶的人α-N-乙酰乳糖胺酶的重组。正在进行的晶体质量的优化正在进行中，以阐明该酶的结构。较少的