Limited proteolysis and reduction-carboxymethylation of rye seed chitinase-a: Role of the chitin-binding domain in its chitinase action

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 Cysl5 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.