Modification of tryptophan residues of plant class III chitinases involved in enzyme activity

Chemical modification of tulip bulb chitinase-1 (TBC-1) and pokeweed leaf chitinase (PLC-B) with N-bromosuccinimide (NBS) suggested the involvement of tryptophan residue (s) in the activity. In the case of TBC-1, at pH 4.0, about 1 mol of tryptophan residues out of 4 mol was oxidized with 5 mol of NBS per mol of TBC-1 and all 4 mol of tryptophan residues were oxidized with 15mol of NBS. At pH 4.5, about 3 mol of tryptophan were finally oxidized although the oxidation rate of the first tryptophan residue was same as at pH 4.0. In both cases, the oxidation of 1 mol of tryptophan residues caused 85% activity loss. Analysis of the oxytryptophan-containing peptides afforded to identify that Trp172 and Trp255 were those which reacted first with NBS at pH 4.0. When PLC-B was reacted with 16-folds molar of NBS at pH 4.0, all seven tryptophan residues were oxidized with a concomitant decrease of chitinase activity. Oxidized tryptophan residues were also determined as those at positions of 165 and 256 in PLC-B. From these results, it was inferred that the rapidly oxidized tryptophan residues in plant class HI chhitinases were involved in their chitinase activity.