Microbial carboxyl proteinases related to a fatal neurodegenerative disease: proposal for a novel catalytic mechanism

与致命性神经退行性疾病相关的微生物羧基蛋白酶：提出一种新的催化机制

基本信息

批准号：
13460043
负责人：
ODA Kohei
金额：
$ 8.45万
依托单位：
KYOTO INSTITUTE OF TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

We carrited out enzymatic and molecular biological investigations on microbial pepstatin-insensitive carboxyl proteinases (PSCP, XSCP, kumamolisin, and J-4) and their mammalian homologue, the CLN2 gene product (CLN2 protein), which is related to the fatal neurodegenerative disease. Compared these results with the data from structural analyses of PSCP and kumamolisin, we clarified their structures and novel catalytic mechanism and discussed on their molecular evolution, too.SUMMARY IN 2001:(i) Conserved serine residuesThe conserved serine residues among five enzymes (PSCP, XSCP, kumamolisin, J-4, and the CLN2 protein) were elucidated to be involved in the catalytic residues by their site-directed mutagenesis studies.(ii) Structural analysis of PSCP and its catalytic mechanismStructural analysis has revealed that a novel type of catalytic triad composed of Ser287, GIu80, and Asp84 residues is involved in its catalytic function. They were confirmed to be the catalytic residues of PSCP by … More site-directed mutagenesis and inhibitor studies.(iii) New inhibitors for kumamolisin and its applicationNovel type of inhibitors were designed and synthesized, and they were used for structural analysis of kumamolisin.SUMMARY IN 2002:(i) Structural analysis of prepro-PSCPS287A mutant was expressed as the insoluble prepro-protein, and the refolded enzyme did not show any autocatalytic processing- and proteinase-activity. These results strongly suggested that Ser287 residue was involved in the catalytic residues. Structural analysis of the S287A mutant is now in progress.(ii) Catalytic residues in kumamolisinBased on the structure of kumamolisin, Glu32 and Trp129 residues were suggested to be an unique catalytic residues in kumamolisin molecule. The k_<cat>/K_m values of the E32A and W129A mutants were about 6 and 4 % of that of the control, indicating that both residues were involved in the catalytic function.(iii) Structural analysis of the CLN2 proteinThe CLN2 protein was elucidated to have a substrate-binding site consisting of six subsites (S3-S3') by kinetics analysis. Structural analysis of the CLN2 protein expressed in silkworm pupae is now in progress. Less

我们对微生物胃酶抑素不敏感的羧基蛋白酶（PSCP、XSCP、kumamolisin 和 J-4）及其哺乳动物同源物 CLN2 基因产物（CLN2 蛋白）进行了酶学和分子生物学研究，CLN2 基因产物与致命的神经退行性疾病有关。将这些结果与 PSCP 和 kumamolisin 的结构分析数据进行比较，我们阐明了它们的结构和新的催化机制，并讨论了它们的分子2001 年总结：(i) 保守丝氨酸残基五种酶（PSCP、XSCP、kumamolisin、J-4 和 CLN2 蛋白）中的保守丝氨酸残基被阐明通过其定点参与催化残基(ii) PSCP 的结构分析及其催化机制结构分析揭示了一种新型的由Ser287、GIu80和Asp84残基组成的催化三联体参与了其催化功能。通过定点诱变和抑制剂研究，证实它们是PSCP的催化残基。(iii)新型kumamolisin抑制剂及其应用新型类型。设计并合成了多种抑制剂，并将其用于 kumamolisin 的结构分析。 2002：(i) 强前原 PSCPS287A 突变体的结构分析被表达为不溶性前原蛋白，并且重折叠酶没有显示任何自催化加工和蛋白酶活性。这些结果表明 Ser287 残基参与催化残基。 S287A突变体的结构分析正在进行中。(ii) kumamolisin中的催化残基基于结构kumamolisin、Glu32 和 Trp129 残基被认为是 kumamolisin 分子中独特的催化残基，E32A 和 W129A 突变体的 k_<cat>/K_m 值分别约为对照的 6 % 和 4 %，表明两者均有效。 (iii) CLN2蛋白的结构分析 CLN2蛋白为通过动力学分析阐明其具有由六个亚位点 (S3-S3') 组成的底物结合位点目前正在对蚕蛹中表达的 CLN2 蛋白进行结构分析。