IDENTIFICATION OF THE TRYPTOPHAN RESIDUE LOCATED AT THE SUBSTRATE-BINDING SITE OF RYE SEED CHITINASE-E

Chemical modifications of rye seed chitinase-c (RSC-c) with various re agents suggested the involvements of tryptophan and glutamic/aspartic acid residues in the activity, Of these, the modification of tryptopha n residues with N-bromosuccinimide (NBS) was investigated in detail. I n the NBS-oxidation at pH 4.0, two of the six tryptophan residues in R SC-c were rapidly oxidized and the chitinase activity was almost compl etely lost. On the other hand, in the NBS-oxidation at pH 5.9, only on e tryptophan residue was oxidized and the activity was greatly reduced , Analyses of the oxidized tryptophan-containing peptides from the try ptic and chymotryptic digests of the modified RSC-c showed that two tr yptophan residues oxidized at pH 4.0 are Trp72 and Trp82, and that oxi dized at pH 5.9 is Trp72. The NBS-oxidation of Trp72 at pH 5.9 was pro tected by a tetramer of N-acetylglucosamine (NAG(4)), a very slowly re active substrate for RSC-c, and the activity was almost fully retained , In the presence of NAG(4), RSC-c exhibited an UV-difference spectrum with maxima at 284 nm and 293 nm, attributed to the red shift of the tryptophan residue, as well as a small trough around 300 nm probably d ue 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 involved in the binding to substrate.