Discovery of two novelproteinases andtheirbi rl emical resemph

Sedolisin：1)CLN2亚基的结构分析：设计合成了组合底物文库，亚基分析正在进行中。2)CLN_2的结构分析：研究了CLN_2的表达和纯化方法，建立了一种高效的CLN_2纯化方法。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转变为Ala，其反应动力学表明这些残基为催化残基。在利用组合文库对底物特异性进行详细分析的基础上，开发了合成抑制剂(Y.Kataoka等人：FEBSE Letters，579,2991-2994(2005))。此外，还制备了酶-抑制剂复合体，结构分析的结果揭示了反应机理(B.Pillai，et.J.Mol Biol 365,343-361(2007))。2)自然界分布：未完成。3)开发安全的农用化学品：相继开发出抑制常数在亚纳米摩尔水平的多肽抑制剂。该抑制剂对植物病原微生物的效果将在实验室规模上确定。

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.