Identification of a Catalytic Residue of Clostridium paraputrificum N-Acetyl-β-D-glucosaminidase Nag3A by Site-Directed Mutagenesis

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概要

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• Clostridium paraputrificum M-21 β-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 Km 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.

• 社団法人 日本農芸化学会の論文
• 2006-05-23

著者

• Ohmiya K

  Graduate School Of Bioresources Mie Univ.

• Ohmiya Kunio

  Faculty Of Bioresources Mie University

• KIMURA Tetsuya

  Faculty of Bioresources, Mie University

• Kajino T

  Special Research Laboratory Ii Toyota Central R&d Laboratories Inc.

• MIYAKE Hideo

  Faculty of Fisheries, Hokkaido University

• Kimura Tetsuya

  National Center For Seeds And Seedlings

• SAKKA Kazuo

  Faculty of Bioresources, Mie University

• Miyake Hideo

  Faculty Of Fisheries Hokkaido University

• Sakka K

  Graduate School Of Bioresources Mie Univ.

• LI Huazhong

  School of Medicine, Southern Yangtze University

• ZHAO Guangshan

  Faculty of Bioresources, Mie University

• UMEKAWA Hayato

  Faculty of Bioresources, Mie University

• Umekawa Hayato

  Faculty Of Bioresources Mie University

• Li Huazhong

  School Of Medicine Southern Yangtze University

• Sakka Kazuo

  Faculty Of Bioresources Mie University

• Zhao Guiwen

  Department Of Chemistry University Of Science And Technology Of China

• Karita S

  Graduate School Of Regional Innovation Studies Mie Univ.

• Kimura Tetsuya

  Graduate School Of Bioresources Mie University

• Miyake Hideo

  Faculty Of Bioresources Mie University

• Kimura Tetsuya

  Faculty Of Bioresources Mie University

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