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-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.
Yamagami
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Laboratory of Protein Chemistry & Engineering, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
Funatsu
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G
D000117:Acetylglucosamine / Q000737:chemistry; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D002688:Chitinases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry; D008969:Molecular Sequence Data; D012434:Secale / Q000201:enzymology*; D012639:Seeds / Q000201:enzymology*; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry
D003432:Cross-Linking Reagents; D001224:Aspartic Acid; C022010:glycine ethyl ester; D002688:Chitinases; D002918:Chymotrypsin; D014357:Trypsin; D005022:Ethyldimethylaminopropyl Carbodiimide; D005998:Glycine; D000117:Acetylglucosamine