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-) d omain (48 residues) and a catalytic (Cat-) domain (254 residues), The hydrolytic activity of the isolated Cat-domain toward soluble glycolch itin, was similar to that of RSC-a, but that toward insoluble colloida l chitin was 28% of that of RSC-a, Five disulfide bonds in the CB-doma in were reduced with 2-mercaptoethanol (2-ME) in the absence of denatu ring agents by an ''all-or-none'' process, that is, once the disulfide bond between Cys15 and Cys42 in the CB-domain was cleaved, the remain ing four disulfide bonds were reduced very easily, The reduced and car boxymethylated RSC-a completely lost the chitin-binding ability, but r etained 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 towa rd insoluble chitin derivatives by binding to them.