Novel Carboxyl Proteinases : Structure, Function, and Evolution

对假单胞菌101号CP(PCP)、Xanthomonas sp.CP(XCP)、凝结芽孢杆菌CP(J-4)、新芽孢杆菌等进行了“胃抑素不敏感CPS”的系列研究。MN-32(库曼溶血素)和人类来源的CLN 2。取得了以下研究成果：1.初步结构我们成功地克隆了J-4蛋白酶基因。上述“胃抑素不敏感CPS”的所有初级序列均被完全测定。分析了枯草杆菌溶血素的底物特异性。结果表明，该酶的S2亚基很小，S2‘亚基由亲水性氨基酸残基和其他细菌来源的胃抑素不敏感CP组成。利用定点突变技术对CLN 2和4种细菌CP的催化氨基酸残基进行了分析，特别是对丝氨酸残基进行了分析。结果表明，这些CPS的催化残基主要由天冬氨酸、谷氨酸和丝氨酸残基组成，并存在一些有待解决的问题。阐明了五氯苯酚的三维结构(《自然结构生物学》，2001年5月出版)。(L)PCP的三维结构与一种典型的丝氨酸蛋白酶--枯草杆菌毒素BPN‘基本相似。(2)催化残基由Asp84、Glu80和Ser287组成。目前还没有关于CPS具有丝氨酸残基参与其催化作用的报道。因此，从遗传学和结构的角度，这些CPS被阐明为一种新的类型的蛋白酶。2000年11月10日，我们组织了一次名为“KIT国际会议”的国际会议，命名为“丝氨酸-羧基蛋白酶及其抑制剂”。

A series of research for "pepstatin-insensitive CPs" was carried out for Pseudomonas sp.No.101 CP (PCP), Xanthomonas sp.CP (XCP), Bacillus coagulans CP (J-4), Bacillus novosp. MN-32 (kumanolysin), and CLN 2 of the human origin. The following results were obtained.1. Primary structures We succeeded for cloning of J-4 proteinase gene. All of the primary sequences of "pepstatin-insensitive CPs" as described above were completely determined.2. Subsite structures A substrate specificity of kumamolysin was analyzed. It was clarified that S2 subsite of the enzyme was very small, and S2' subsite was composed of hydrophilic aminoacid residues as well as other pepstatin-insensitive CPs of bacterial origin.3. Catalytic residues The presumed catalytic amino acid residues of CLN2 and 4-types of bacterial CPs were analyzed by site-directed mutagenesis, especially focused on Ser residue. As a result, catalytic residues of these CPs were elucidated to be composed of Asp, Glu, and Ser residues, beside of some unclarified problems.4. Three-dimensional structure Three-dimensional structure of PCP was elucidated (Nature Structural Biology, in May, 2001 in press). (l) Three-dimensional structure of PCP was basically similar to a typical serine proteinase, subtilisin BPN'. (2) The catalytic residues were proved to be composed of Asp84, Glu80, and Ser287. There are no reports about CPs that have Ser residues involved in their catalysis. Therefore, these CPs were elucidated to be a new type of proteinase based on genetical and structural approach. We ranked these CPs as a fifth proteinase family, and named it as "serine-carboxyl proteinase"On Nov.10 in 2000 year, we organized an international conference named as "KIT International Conference on Carboxyl Proteinases and Their Inhibitors".