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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

我们对微生物胃蛋白酶抑制剂不敏感的羧基蛋白酶（PSCP、XSCP、β-amolisin和J-4）及其哺乳动物同源物CLN 2基因产物（CLN 2蛋白）进行了酶学和分子生物学研究，CLN 2基因产物与致命的神经退行性疾病有关。2001年：（i）保守的丝氨酸残基通过定点突变研究，发现了5种酶（PSCP、XSCP、CXCP、CXCP、J-4和CLN 2蛋白）中保守的丝氨酸残基参与了催化残基的形成。(ii)PSCP的结构分析及其催化机理结构分析表明，PSCP的催化功能涉及一种由Ser 287、Glu 80和Asp 84残基组成的新型催化三联体。经质谱分析，证实它们是PSCP的催化残基。 ...更多信息定点诱变和抑制剂研究。(iii)2002年综述：（i）前原PSCPS 287 A突变体的结构分析结果表明，该突变体表达的是不溶性的前原蛋白，复性后的酶不显示任何自催化加工和蛋白酶活性。这些结果强烈地表明Ser 287残基参与了催化残基。S287 A突变体的结构分析目前正在进行中。(ii)从结构上分析，Glu 32和Trp 129残基可能是氯霉素分子中唯一的催化残基。E32 <cat>A和W129 A突变体的k/Km值分别为对照的6%和4%，表明两个残基都参与了催化功能。(iii)CLN 2蛋白的结构分析通过动力学分析，阐明CLN 2蛋白具有由六个亚位点（S3-S3 '）组成的底物结合位点。对家蚕蛹中表达的CLN 2蛋白的结构分析正在进行中。少