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¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1993 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1992 : ¥1,600,000 (Direct Cost : ¥1,600,000)
It is well known that carboxyl proteinases are commonly inhibited by pepstain^<1)>, DAN^<2)>, and EPNP^<3)>, and their catalytic residues are composed of two aspartic acid residues. Thus, carboxyl proteinases are termed aspartic proteinases. These enzymes are highly homologous in both the primary and tertiary structures.
We have isolated novel carboxyl proteinases from frugi, bacteria and also thermophilic bacteria based on their insensitivities to pepstatin, DAN and EPNP.These enzymes were tentatively named pepstatin-insensitve carboxyl proteinases. In one of our studies, the primary structure of carboxyl proteinase B (consisting of 204 amino acids) from a fungus Scytalidum lignicolum has been established, and one of the catalytic residues of the enzyme was clarified to be Glu-53. This is the first report on glutamic proteinase. It seemed probable that the pepstain-insensitive carboxyl proteinases are not aspartic proteinases but glutamic proteinases. To confirm this possibility, we fo
cussed our studies on a pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. No. 101(PCP), which is the first carboxyl proteinase isolated from prokaryote cells. The primary structure of PCP has been determined to be a single polypeptide composed of 372 amino acid residues with one disulfide bridge. PCP does not have any homologous structure to those of aspartic proteinases reported so far. Moreover, the well-conserved structure, -Asp^<**>-Thr-Gly-(Asp^<**> : catalytic residue) in the active center of aspartic proteinases was not observed.
In this study, the following results wera obtained.
1. Identification of Catalytic Residues In our attempt to use inhibitor in the study of active center, we had isolated a novel inhibitor. tyrostatin (N-isovaleryl-tyrosyl-leucyl-tyrosinal, Ki = 2.5Nm) from Kitasatosporia sp.No.55. Based on the cmemical structure. we succeeded in synthesizing a compeptive inhibitor, available for probing the catalytic residues of PCP(N-benzyloxycarbonyl-L-phenyl-atanine-2,3-epoxypropyl ester).
2. Analysis of PCP Gene We determined the whole DNA sequence of the PCP gene(about 3 Kbp). It was elucidated that PCP is composed of prepro part protein (215 amino acid residues) and mature protein (372 amino acid residues). Primary structure of the mature protein was identical to that chemically determined previouly. It was suggested that the propart protein plays important roles in the activation as well as secretion through the double layr of the cell.
Accordingly, it is ready now to study the structure-function relationships, especially the catalytic residues on both side of protein and DNA level.
1) pepstatin, pepsin inhibitor ; 2) DAN, diazoacetyl-DL-norleucine methylester ; 3) EPNP, 1,2-epoxy-3-(p-nitrophenoxy) propane. Less