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Discovery of two novelproteinases andtheirbi rl emical resemph

两种新型蛋白酶的发现及其生物学研究

基本信息

批准号：
18380068
负责人：
HIRAGA Kazumi
金额：
$ 9.38万
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：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

Sedolisin : 1) Structural analysis of CLN2 subsite : Combinatorial substrate library was designed and synthesized, and subsite-analysis is underway. 2) structure analysis of CLN2 : Procedures for expression and purification were investigated and an extremely efficient purification method was constructed. Large amount of CLN2 was prepared and the crystallization of CLN2 is undergoing.Eqolisin family: 1) Elucidation of reaction mechanism: Glu136 and G1n53, presumed catalytic residues by structural analysis, were changed to Ala and their reaction kinetics revealed that these residues were the catalytic resideues. Based on the detailed analysis for substrate specificity using combinatorial library, synthetic inhibitor was developed (Y. Kataoka et al: FEBSE Letters, 579, 2991-2994 (2005)). Furthermore, enzyme-inhibitor complex was prepared and the obtained results from the structural analysis revealed the reaction mechanism (B. Pillai, et. al.: J. Mol Biol 365, 343-361 (2007)). 2) Distribution in nature: not completed. 3) Development of safety agrichemicals: Peptide inhibitors having inhibitory constant at subnano-molar-level were successively developed. The effect of the inhibitor for phytopathogenic microbials will be determined at lab scale.

Sedolisin： 1) CLN2亚位点的结构分析：设计并合成了组合底物库，正在进行亚位点分析。 2) CLN2的结构分析：研究了表达和纯化程序并构建了极其有效的纯化方法。大量的CLN2被制备出来，并且CLN2的结晶正在进行中。 Eqolisin家族： 1)反应机理的阐明：结构分析推测的催化残基Glu136和G1n53被转变为Ala，它们的反应动力学表明这些残基是催化残基。基于使用组合文库对底物特异性的详细分析，开发了合成抑制剂（Y. Kataoka 等人：FEBSE Letters, 579, 2991-2994 (2005)）。此外，制备了酶-抑制剂复合物，并且从结构分析获得的结果揭示了反应机制(B.Pillai等人：J.Mol Biol 365, 343-361(2007))。 2) 分配性质：未完成。 3）安全农药开发：陆续开发出抑制常数为亚纳摩尔水平的肽抑制剂。抑制剂对植物病原微生物的作用将在实验室规模上确定。