| Budget Amount *help |
¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
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".
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