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.
Sokolova
E A
EA
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia.
Starkova
N N
NN
Vorotyntseva
T I
TI
Zamolodchikova
T S
TS
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