IDENTIFICATION OF THE ASPARTIC-ACID RESIDUE LOCATED AT OR NEAR SUBSTRATE-BINDING SITE OF RYE SEED CHITINASE-C

Carboxyl groups of rye seed chitinase-c (RSC-c) were modified with 1-e thyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine ethyl est er (GEE) at pH 5.5 and 5 degrees C in the presence and absence of (Glc NAc)(4). In the absence of (GlcNAc)(4), 5.2 carboxyl groups were modif ied by 90 min-reaction and the chitinase activity was reduced to 2.0%, while in the presence of (GlcNAc)(4), 4.6 carboxyl groups were modifi ed and 72% of the activity was retained. To identify the carboxyl grou p protected by (GlcNAc), from the modification, RSC-c was first modifi ed with EDC and GEE in the presence of (GlcNAc)(4) and then radiolabel ed with EDC and [C-14]GEE in the absence of (GlcNAc)(4). Analyses of t he radioactive peptides from the tryptic and chymotryptic digests of r adiolabeled RSC-c showed that the main radiolabeled carboxyl group is that of Asp95, suggesting that Asp95 is located at or near substrate-b inding site of RSC-c.