Li Huazhong, Zhao Guangshan, Miyake Hideo, Umekawa Hayato, Kimura Tetsuya, Ohmiya Kunio, Sakka Kazuo
School of Medicine, Southern Yangtze University, Wuxi, China.
Biosci Biotechnol Biochem. 2006 May;70(5):1127-33. doi: 10.1271/bbb.70.1127.
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.
副腐败梭菌M-21β-N-乙酰氨基葡萄糖苷酶3A（Nag3A）是一种归类于糖苷水解酶家族3的酶。为了鉴定该酶的催化残基，将突变引入高度保守的Glu和Asp残基。用Ala取代Asp175消除了催化活性，而圆二色光谱没有变化，这强烈表明该残基是催化残基、亲核试剂/碱或质子供体。由于突变酶D119N、D229N、D229A和D274N的K(m)值与野生型酶相比增加了17至41倍，Asp119、Asp229和Asp274似乎参与底物识别和结合。考虑到先前的研究，我们推测Asp303是副腐败梭菌Nag3A的催化亲核试剂，Asp175是质子供体。
