Aorsin, a novel serine proteinase with trypsin-like specificity at acidic pH.
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 pH 4.0. The specificity of aorsin appeared to require a basic residue at the P(1) 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 k (cat)/ K(m) values of the mutant enzymes Glu(86)-->Gln, Asp(211)-->Asn and Ser(354)-->Thr were 3-4 orders of magnitude lower and Asp(90)-->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.
Lee
Byung Rho
BR
Laboratory of Molecular Enzymology, Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan. brlee@t.soka.ac.jp
Furukawa
Masato
M
Yamashita
Koichiro
K
Kanasugi
Yurie
Y
Kawabata
Choko
C
Hirano
Kenichi
K
Ando
Kenichi
K
Ichishima
Eiji
E
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology*; D001483:Base Sequence; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D014357:Trypsin