title: Limited proteolysis and reduction-carboxymethylation of rye seed chitinase-a: role of the chitin-binding domain in its chitinase action.
abstract: By a limited proteolysis with thermolysin, rye seed chitinase-a (RSC-a) was separated into a N-terminal cysteine-rich chitin-binding (CB-) domain (48 residues) and a catalytic (Cat-) domain (254 residues). The hydrolytic activity of the isolated Cat-domain toward soluble glycolchitin, was similar to that of RSC-a, but that toward insoluble colloidal chitin was 28% of that of RSC-a. Five disulfide bonds in the CB-domain were reduced with 2-mercaptoethanol (2-ME) in the absence of denaturing agents by an "all-or-none" process, that is, once the disulfide bond between Cys15 and Cys42 in the CB-domain was cleaved, the remaining four disulfide bonds were reduced very easily. The reduced and carboxymethylated RSC-a completely lost the chitin-binding ability, but retained 50% of the hydrolytic activity toward colloidal chitin of RSC-a. From these results, it was shown that RSC-a consists of a CB-domain and a Cat-domain connected by a flexible linker, and it was suggested that the CB-domain increases the hydrolytic action of Cat-domain toward insoluble chitin derivatives by binding to them.
authors.affiliation: Laboratory of Protein Chemistry & Engineering, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Yamagami
authors.forename: G
authors.initials: G
authors.lastname: Funatsu
mesh_terms: D000595:Amino Acid Sequence; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008745:Methylation; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification; D012434:Secale / Q000201:enzymology* / Q000378:metabolism; D012639:Seeds / Q000201:enzymology / Q000378:metabolism; D013820:Thermolysin; D014357:Trypsin
chemical_list: D010455:Peptides; D002686:Chitin; D002688:Chitinases; D014357:Trypsin; D013820:Thermolysin