A serine protease from the bovine duodenal mucosa, chymotrypsin-like duodenase.
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)/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. Alpha-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.
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia.
E A
EA
Sokolova
N N
NN
Starkova
T I
TI
Vorotyntseva
T S
TS
Zamolodchikova
D000595:Amino Acid Sequence; D000818:Animals; D056649:Cathepsin G; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism; D004386:Duodenum; D007413:Intestinal Mucosa / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D014462:Ultrafiltration
D010446:Peptide Fragments; D002403:Cathepsins; D012697:Serine Endopeptidases; C078261:duodenase; D002918:Chymotrypsin; D056649:Cathepsin G