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Studies on Carboxyl Proteinases : Comparative Analysis of Functional Structures and Catalytic Mechanisms of Proctases A and B

羧基蛋白酶的研究：蛋白酶A和B的功能结构和催化机制的比较分析

基本信息

批准号：
05453209
负责人：
TAKAHASHI Kenji
金额：
$ 4.8万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

1.By specific chemical modifications, carboxyl groups were shoun to be involved in the active site of Proctase A as in the case of Proctase B.Asp14 and Asp 111 in the heavyc hain of Proctase A were indicated to be the catalytic groups by site-directed mutagenesis studies using an E.coli expression system for pro-Proctase A.2.A cDNA of prepro-Proctase B was cloned and sequenced, giving the complete amino acid sequence of the preproenzyme. Pro-Proctase B was shown to be autocatalytically activated under acidic conditions. Site-direcited mutagenesis studies on pro-Proctase B revealed that Lys 36 and Arg 32 are important for the shroctural stability of the proform and its activation, respectively.3.The expression system of pro-Proctase A in E.coli was established, and the proenzyme was shown to be autocatalytically activated under acidic conditions like pro-Proctase B.4.The profiles of denaturation of Proctase A at different pHs and temperatures were in vestigated using various physico-chemical methods in clading NMR and circular dichroism, which revealed unique denaturation profile of the enzyine.5.Nearly all proton signals in NMR of the light chain of Proctase A were assigned and the three-dcmensional structure of the chain was determined. Further, a method for preparation of isotopically labeled Proctase A using Aspergillus niger was established, and using these ^<15>N and/or ^<13>C-labeled Protase A,the three-dimensional structure was investigated.6.A unique substrate specificity of Proctase A was elucidatid using oxidized B chain of insulin ets, which in as quite differant from that of Proctase B.In addition, the interaction of an inhibitor peptide with Proctase A was investigated by NMR.7.Isomorphous metal (Pt and Hg)derivatives of Proctase A were crystallized and the three-dimensional shructure of the enzyrne was partly elarified by X-ray crystallographic analysis.

1.通过特定的化学修饰，发现Proctase A的活性位点与Proctase b一样有羧基参与。利用大肠杆菌原Proctase A.2的表达系统进行定点诱变研究，发现Proctase A的重c链中的asp14和Asp 111是催化基团。克隆了原proctase B的cDNA序列，得到了原proctase B的完整氨基酸序列。原proctase B在酸性条件下具有自催化活性。对proproctase B的定点诱变研究表明，Lys 36和Arg 32分别对protase B的结构稳定性和激活起着重要作用。建立了原proctase A在大肠杆菌中的表达体系，结果表明，该原酶与原proctase B.4一样，在酸性条件下具有自催化活性。采用核磁共振和圆二色分析等多种物理化学方法研究了Proctase A在不同ph值和温度下的变性谱，揭示了该酶独特的变性谱。对Proctase A轻链的几乎所有质子信号进行了核磁共振赋值，并确定了轻链的三维结构。在此基础上，建立了用黑曲霉制备同位素标记的蛋白酶a的方法，并利用这些^<15>N和/或^<13> c标记的蛋白酶a进行了三维结构研究。利用胰岛素链的氧化B链阐明了Proctase A独特的底物特异性，这与Proctase B完全不同。此外，NMR.7还研究了一种抑制剂肽与Proctase A的相互作用。对Proctase A的同构金属（Pt和Hg）衍生物进行了结晶，并通过x射线晶体分析部分阐明了该酶的三维结构。