title: Involvements of Trp23 in the Chitin-binding and of Trp131 in the Chitinase Activity of Rye Seed Chitinase-a.
abstract: The oxidation of the tryptophan residues of rye seed chitinase-a (RSC-a) and its isolated catalytic (Cat) domain by N-bromosuccinimide (NBS) was investigated in the absence and presence of oligomers of N-acetylglucosamine (GlcNAc)n. Based on the reactivity toward NBS at pH 5.9, the seven tryptophan residues present in RSC-a are grouped into highly reactive (HR-), low reactive (LR-), and unreactive residues. Analyses of the peptides from 1 tryptophan- and 3 tryptophan-oxidized RSC-a showed that the HR-residue is Trp23 and the LR-residues are Trp131 and Trp141. The chitin-binding ability of RSC-a was lost upon the NBS oxidation of Trp23 at pH 5.9 or pH 7.0. This oxidation was prevented by (GlcNAc)3, which induced a high UV-difference spectrum with maxima at 284 and 293 nm. On the other hand, the chitinase activity of the Cat domain was greatly reduced by the NBS oxidation of Trp131 and Trp141 at pH 5.9, while in the NBS oxidation at pH 6.4, approximately one tryptophan residue was oxidized and about half of the activity was retained. The NBS oxidation of the isolated Cat domain at pH 5.9 was protected by (GlcNAc)4, which induced a UV-difference spectrum with maxima at 284 nm and 293 nm as well as a small trough around 300 nm, similar to that observed in RSC-c. From these results and the previous result that Trp72 in RSC-c is involved in the substrate-binding, it was suggested that Trp23 is highly exposed on the surface of the RSC-a molecule and involved in the chitin-binding, while Trp131 is involved in substrate-binding in its enzyme action.
authors.affiliation: a Laboratory of Protein Chemistry & Engineering, Faculty of Agriculture , Kyushu University , Fukuoka 812-81 , Japan.
authors.forename: T
authors.initials: T
authors.lastname: Yamagami
authors.affiliation: a Laboratory of Protein Chemistry & Engineering, Faculty of Agriculture , Kyushu University , Fukuoka 812-81 , Japan.
authors.forename: G
authors.initials: G
authors.lastname: Funatsu
keywords: N-bromosuccinimide oxidation; active site of enzyme; chitin-binding protein; chitinase; rye seeds