A proteinase that hydrolyses clupeine and salmine at acidic pH, called aorsin, was found in the fungus Aspergillus oryzae. Purified aorsin also hydrolysed benzyloxycarbonyl-Arg-Arg-4-methylcoumaryl-7-amide optimally at pH4.0. The specificity of aorsin appeared to require a basic residue at the P1 position and to prefer paired basic residues. Aorsin activated plasminogen and converted trypsinogen to trypsin. The trypsin-like activity was inhibited strongly by antipain or leupeptin, but was not inhibited by any other standard inhibitors of peptidases. To identify the catalytic residues of aorsin, a gene was cloned and an expression system was established. The predicted mature protein of aorsin was 35% identical with the classical late-infantile neuronal ceroid lipofuscinosis protein CLN2p and was 24% identical with Pseudomonas serine-carboxyl proteinase, both of which are pepstatin-insensitive carboxyl proteinases. Several putative catalytic residues were mutated. The kcat/Km values of the mutant enzymes Glu86→Gln, Asp211→Asn and Ser354→Thr were 3–4 orders of magnitude lower and Asp90→Asn was 21-fold lower than that of wild-type aorsin, indicating that the positions are important for catalysis. Aorsin is another of the S53 family serine-carboxyl proteinases that are not inhibited by pepstatin.
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April 2003
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Research Article|
April 15 2003
Aorsin, a novel serine proteinase with trypsin-like specificity at acidic pH
Byung Rho LEE;
Byung Rho LEE
1
∗Laboratory of Molecular Enzymology, Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan
1To whom correspondence should be addressed (e-mail brlee@t.soka.ac.jp).
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Masato FURUKAWA;
Masato FURUKAWA
†Laboratory of Molecular Enzymology, Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan
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Koichiro YAMASHITA;
Koichiro YAMASHITA
∗Laboratory of Molecular Enzymology, Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan
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Yurie KANASUGI;
Yurie KANASUGI
∗Laboratory of Molecular Enzymology, Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan
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Choko KAWABATA;
Choko KAWABATA
‡Technical Research Center of T. Hasegawa Co., Ltd., Kariyado, 335 Nakahara-ku, Kawasaki 211-0022, Japan
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Kenichi HIRANO;
Kenichi HIRANO
§Gifu R and D Center of Amano Enzyme Inc., 4-179-35, Sue-cho, Kakamigahara, Gifu 509-0108, Japan
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Kenichi ANDO;
Kenichi ANDO
§Gifu R and D Center of Amano Enzyme Inc., 4-179-35, Sue-cho, Kakamigahara, Gifu 509-0108, Japan
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Eiji ICHISHIMA
Eiji ICHISHIMA
∗Laboratory of Molecular Enzymology, Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan
†Laboratory of Molecular Enzymology, Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan
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Publisher: Portland Press Ltd
Received:
October 30 2002
Revision Received:
December 18 2002
Accepted:
January 08 2003
Accepted Manuscript online:
January 08 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 371 (2): 541–548.
Article history
Received:
October 30 2002
Revision Received:
December 18 2002
Accepted:
January 08 2003
Accepted Manuscript online:
January 08 2003
Citation
Byung Rho LEE, Masato FURUKAWA, Koichiro YAMASHITA, Yurie KANASUGI, Choko KAWABATA, Kenichi HIRANO, Kenichi ANDO, Eiji ICHISHIMA; Aorsin, a novel serine proteinase with trypsin-like specificity at acidic pH. Biochem J 15 April 2003; 371 (2): 541–548. doi: https://doi.org/10.1042/bj20021691
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