| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1989: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1988: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1987: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
It is well known that carboxyl proteases are commonly inhibited by pepstatin^<1)> , DAN^<2)> and EPNP^<3)> , and their catalytic residues are composed of two aspartic residues. Thus, carboxyl proteases are termed aspartic proteases. These enzymes are highly homologous in both the primary and tertiary structures.
We have isolated novel carboxyl proteases from fungi, bacteria and also thermophilic bacteria based on their insensitivities to pepstatin, DAN and EPNP. These enzymes were tentatively named pepstatin-insensitve carboxyl proteases. In one of our studies, the primary structure of carboxyl protease B ( consisting of 204 aminb acids) from a fungus Scytalidium lignicolum has been established, one of the catalytic residues of which was clarified to be Glu-53. This is the first report on glutamic protease.
It seemed probable that the pepstatin-insensitive carboxyl proteases are not aspartic proteases but glutamic proteases. To confirm this possibility, we studied a pepstatin-insensitive
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carboxyl protease from Pseudomonas sp. No. 101, which is the the first carboxyl protease isolated from prokaryote cells. We determined the primary structure of the enzyme as a single polypeptide composed of 363 amino acid residues with one disulfide bridge. The elucidated primary structure does not have any homologous structure to those of aspartic proteases reported so far. Moreover, the well-conserved structure, -Asp*-Thr-Gly- in the active center of aspartic proteases was not observed.
In our attempt to use inhibitor in the study of active center we isolated a novel inhibitor from Kitasatosporia sp. No. 55. It was identified as N-isovaleryl-tyrosyl-leucyl-tyrosinal and named it tyrostatin. Tyrostatin was found to inhibit stoichiometrically pepstatin- insensitive carboxyl proteinases of Pseuomonas sp. No. 101.
Further study on identification of the catalytic residues of Pseudomonas carboxyl protease using tyrostatin or its derivatives is now in progress. 1 ) pepstatin, pepsin inhibitor; 2) DAN, diazoacetyl-DL-norleucine methylester; 3) EPNP, 1,2-epou-3-(p- nitrophenoxy) propane. Less
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