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

Takeshi Yamagami, Gunki Funatsu

Research output: Contribution to journalArticle

27 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)1081-1086
Number of pages6
JournalBioscience, Biotechnology and Biochemistry
Volume60
Issue number7
DOIs
Publication statusPublished - Jan 1 1996

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Proteolysis
Chitinases
Chitin
Seed
Seeds
Catalytic Domain
Disulfides
Thermolysin
Mercaptoethanol
Cysteine
Secale
Derivatives

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

Cite this

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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 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.",
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AB - 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.

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