Open AccessA serine protease from the bovine duodenal mucosa, chymotrypsin‐like duodenase
Author(s) -
Sokolova Elena A.,
Starkova Natalia N.,
Vorotyntseva Tatyana I.,
Zamolodchikova Tatyana S.
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2550501.x
Subject(s) - chymotrypsin , chemistry , biochemistry , isoelectric point , serine protease , enzyme , protease , serine , proteases , cathepsin l , active site , trypsin
Chymotrypsin‐like duodenase (ChlD), a new protease from the bovine duodenum mucosa was isolated and purified. The enzyme molecule is a single chain (25 kDa); the native enzyme is a monomer with an isoelectric point > 10.0. ChlD displays the chymotrypsin‐like activity and cleaves 4‐nitroanilide substrates of chymotrypsin, chymases and cathepsin G. ChlD hydrolyzes its best substrate 2‐ N ‐succinylvalylprolylphenylalanine 4‐nitroanilide with k cat of 2.8 s −1 and catalytic efficiency k cat / K m of 2300 M −1 s −1 . The enzyme is stable with a pH range of 3−10 and exhibits the maximum activity at pH 8−10. ChlD is irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulfonyl fluoride, which is indicative of an active‐site serine in this protease. α‐ N ‐tosyl‐ L ‐phenylalanine chloromethane, a specific reagent for a catalytically active His, markedly inhibited ChlD. The enzyme activity was strongly inhibited by several natural inhibitors of serine proteases (from soybean, potato, Lima bean, kidney bean). The N‐terminal sequence of the native ChlD (23 amino acids) shows high similarity, but not identity, to those of duodenase, granzymes, chymases and cathepsin G.