Identification of the aspartic acid residue located at or near substrate-binding site of rye seed chitinase-c

Bioscience, Biotechnology, and Biochemistry
T Yamagami, G Funatsu

Abstract

Carboxyl groups of rye seed chitinase-c (RSC-c) were modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine ethyl ester (GEE) at pH 5.5 and 5 degrees C in the presence and absence of (GlcNAc)4. In the absence of (GlcNAc)4, 5.2 carboxyl groups were modified 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 modified and 72% of the activity was retained. To identify the carboxyl group protected by (GlcNAc)4 from the modification, RSC-c was first modified with EDC and GEE in the presence of (GlcNAc)4 and then radiolabeled with EDC and [14C]GEE in the absence of (GlcNAc)4. Analyses of the radioactive peptides from the tryptic and chymotryptic digests of radiolabeled RSC-c showed that the main radiolabeled carboxyl group is that of Asp95, suggesting that Asp95 is located at or near substrate-binding site of RSC-c.

Citations

Jul 1, 1969·Canadian Journal of Microbiology·J Monreal, E T Reese
Jan 5, 1993·Journal of Molecular Biology·P J HartJ D Robertus

Related Concepts

Aspartic Acid, Magnesium-Potassium (2:1:2) Salt
Endochitinase
Digests
CHIT1
Glycine
Glycine (Plant)
Avazyme
Ligand Binding Domain
Chitinase Activity
Ethyldimethylaminopropyl Carbodiimide

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