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-methyl-coumaryl-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 c a t /K m values of the mutant enzymes Glu 8 6 →G1n, Asp 2 1 1 →Asn and Ser 3 5 4 → Thr were 3-4 orders of magnitude lower and Asp 9 0 →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.