serine protease from the bovine duodenal mucosa, chymotrypsin-like duodenase

• Published in: European journal of biochemistry Vol. 255; no. 2; pp. 501 - 507
• Main Authors: Sokolova, E.A, Starkova, N.N, Vorotyntseva, T.I, Zamolodchikova, T.S
• Format: Journal Article
• Language: English
• Published: Berlin & Heidelberg Springer‐Verlag 15.07.1998
• Subjects: Amino Acid Sequence; amino acid sequences; Animals; Cathepsin G; Cathepsins - chemistry; Cathepsins - metabolism; Cattle; Chromatography, Affinity; Chromatography, Ion Exchange; Chymotrypsin - chemistry; Chymotrypsin - metabolism; comparisons; duodenal mucosa; Duodenum; intestinal mucosa; Intestinal Mucosa - enzymology; Kinetics; Molecular Sequence Data; Peptide Fragments - chemistry; Sequence Alignment; Sequence Homology, Amino Acid; Serine Endopeptidases - chemistry; Serine Endopeptidases - isolation & purification; Serine Endopeptidases - metabolism; serine protease; serine proteinases; specificity; Substrate Specificity; Ultrafiltration
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1046/j.1432-1327.1998.2550501.x

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 kcat of 2.8 s−1 and catalytic efficiency kcat/Km 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.