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タイトル: Novel Mechanism of Enzymatic Hydrolysis Involving Cyanoalanine Intermediate Revealed by Mass Spectrometric Monitoring of an Enzyme Reaction (MOLECULAR BIOFUNCTION-Molecular Microbial Science)
著者: Esaki, Nobuyoshi
Kurihara, Tatsuo  kyouindb  KAKEN_id  orcid https://orcid.org/0000-0002-7777-1378 (unconfirmed)
Li, Yong-Fu
Ichiyama, Susumu
キーワード: 2-Haloacid dehalogenase
Ion-spray mass spectrometry
Cyanoalanine residue
発行日: Mar-2001
出版者: Institute for Chemical Research, Kyoto University
誌名: ICR Annual Report
巻: 7
開始ページ: 46
終了ページ: 47
抄録: L-2-Haloacid dehalogenase from Pseudomonas sp. YL catalyzes the hydrolytic dehalogenation of L-2- haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids. Asp10 of the enzyme functions as a catalytic nucleophile: the residue attacks the -carbon of the substrate to form an ester intermediate, which is subsequently hydrolyzed to release the product. Surprisingly, replacement of Asp10 by Asn did not completely inactivate the enzyme. We found that Asn10 of the D10N mutant enzyme is spontaneously deamidated to yield Asp, though slowly, causing increasing activity of the D10N preparation. We also revealed by mass spectrometric monitoring of the enzyme reaction that the D10N mutant enzyme itself catalyzes the hydrolytic dehalogenation: Asn10 attacks the substrate to form an imidate, and a proton and D-lactic acid are eliminated to produce a nitrile (a -cyanoalanine residue), followed by hydrolysis to reproduce Asn10. This is the first report of the function of Asn to catalyze nucleophilic substitution through its dynamic structural change that includes conversion to a - cyanoalanine residue as an intermediate structure.
URI: http://hdl.handle.net/2433/65267
出現コレクション:Vol.7 (2000)

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