Identification of the Seed Chitinase - c Tryptophan ResidueLocated atthe Substrate - binding Site of Rye

Chemical modifications of rye seed chitinase-c (RSC-c) with yarious reagents suggested the inyolyements of tryptophan and glutamiclaspartic acid residues in the activity. Of these, the modification of tryptophan residues wjth ?V・bromosuccinimide (NBS) was inyestigated in detail. In the NBS-oxidation at pH 4.0, two of the six tryptophan residues in RSC-c were rapidly oxidized and the chitinase activity was almost completely lost. On the other hand, in the NBS-oxidation at pH 5.9, only one tryptophan residue was oxidized and the actiyity was greatly reduced. Analyses of the oxidized tryptophan-containing peptides from the tryptic and chymotryptic digests of the modified RSC-c showed that two tryptophan residues oxidized at pH 4.0 are Trp72 and Trp82, and that oxidized at pH 5.9 is Trp72. The NBS-oxidation of TTp72 at pH 5.9 was protected by a tetramer of iY-acetylglueosamine (NAG.), a very slowly reactiye substrate for RSC-c, and the actiyity was almost fu11y retained. In the presence of NAG4, RSC-c exhibited an UV-diffbrence spectrum with maxima at 284nm and 293 nm, attributed to the red shift of the tryptophan residue, as well as a small trough around 300nm probably due to an alteration of the environment of the tryptophan residue. From these results, it was suggested that Trp72 is exposed on the surface of the RSC-c molecule and inyolyed in the binding to substrate.