title: Kinetic analysis of a chitinase from red sea bream, Pagrus major.
abstract: Kinetic analysis was done on the 46-kDa chitinase (EC 3.2.1.14) purified from the stomach of red sea bream, Pagrus major, using glycolchitin and N-acetylchitooligosaccharides (GlcNAc(n), n=2-6) as substrates. High activity was observed at two pHs, such as 2.5 and 9.0, toward glycolchitin as seen in other insect chitinases, and also at both pH 2.5 and 5.0 even toward a short substrate, N-acetylchitopentasaccharide. Allosamidin competitively inhibited chitinase with Ki value of 0.0214 microM at pH 2.5 and 0.0024 microM at pH 9.0 in the reaction of glycolchitin. Substrate inhibition was observed in the reaction of N-acetylchitopentasaccharide. The anomeric forms of the products from N-acetylchitooligosaccharides were analyzed to be beta anomer by the high pressure liquid chromatography (HPLC) method. The data for both beta-anomer formation and allosamidin inhibition suggest that red sea bream chitinase belongs to family 18 of glycosyl hydrolases. This suggestion is also supported by the results for the N-terminal amino acid sequence.
authors.affiliation: Laboratory of Biochemistry, Department of Biological Science, Faculty of Agriculture, Yamaguchi University.
authors.forename: Shuji
authors.initials: S
authors.lastname: Karasuda
authors.forename: Kosuke
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Michiko
authors.initials: M
authors.lastname: Kono
authors.forename: Shohei
authors.initials: S
authors.lastname: Sakuda
authors.forename: Daizo
authors.initials: D
authors.lastname: Koga
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002686:Chitin / Q000031:analogs & derivatives* / Q000378:metabolism; D002688:Chitinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism; D021541:Sea Bream; D014312:Trisaccharides / Q000494:pharmacology
chemical_list: D009844:Oligosaccharides; D014312:Trisaccharides; C054851:allosamidin; D002686:Chitin; C031610:glycolchitin; D002688:Chitinases; D000117:Acetylglucosamine