title: Identification of a catalytic residue of Clostridium paraputrificum N-acetyl-beta-D-glucosaminidase Nag3A by site-directed mutagenesis.
abstract: Clostridium paraputrificum M-21 beta-N-acetylglucosaminidase 3A (Nag3A) is an enzyme classified in family 3 of the glycoside hydrolases. To identify catalytic residues of this enzyme, mutations were introduced into highly conserved Glu and Asp residues. Replacement of Asp175 with Ala abolished the catalytic activity without change in the circular dichroism spectrum, strongly suggesting that this residue is a catalytic residue, a nucleophile/base or a proton donor. Since the K(m) values of mutant enzymes D119N, D229N, D229A and D274N increased 17 to 41 times as compared with that of wild-type enzyme, Asp119, Asp229, and Asp274 appear to be involved in substrate recognition and binding. Taking previous studies into consideration, we presume that Asp303 is the catalytic nucleophile and Asp175 is the proton donor of C. paraputrificum Nag3A.
authors.affiliation: School of Medicine, Southern Yangtze University, Wuxi, China.
authors.forename: Huazhong
authors.initials: H
authors.lastname: Li
authors.forename: Guangshan
authors.initials: G
authors.lastname: Zhao
authors.forename: Hideo
authors.initials: H
authors.lastname: Miyake
authors.forename: Hayato
authors.initials: H
authors.lastname: Umekawa
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kimura
authors.forename: Kunio
authors.initials: K
authors.lastname: Ohmiya
authors.forename: Kazuo
authors.initials: K
authors.lastname: Sakka
mesh_terms: D000118:Acetylglucosaminidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics*; D002942:Circular Dichroism; D003013:Clostridium / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013379:Substrate Specificity
chemical_list: D001224:Aspartic Acid; D000118:Acetylglucosaminidase