Catalysis-Linked Inactivation of Fluoroacetate Dehalogenase by Ammonia: A Novel Approach to Probe the Active-Site Environment.
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概要
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Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes the hydrolytic dehalogenation of fluoroacetate and other haloacetates. Asp105 of the enzyme acts as a nucleophile to attack the α-carbon of haloacetate to form an ester intermediate, which is subsequently hydrolyzed by a water molecule activated by His272 [Liu, J. Q., Kurihara, T., Ichiyama, S., Miyagi, M., Tsunasawa, S., Kawasaki, H., Soda, K., and Esaki, N. (1998) J. Biol. Chem. 273, 30897-30902]. In this study, we found that FAc-DEX is inactivated con-comitantly with defluorination of fluoroacetate by incubation with ammonia. Mass spectrometric analyses revealed that the inactivation of FAc-DEX is caused by nucleophilic attack of ammonia on the ester intermediate to convert the catalytic residue, Asp105, into an asparagine residue. The results indicate that ammonia reaches the active site of FAc-DEX without losing its nucleophilicity. Analysis of the three-dimensional structure of the enzyme by homology modeling showed that the active site of the enzyme is mainly composed of hydrophobic and basic residues, which are considered to be essential for an ammonia molecule to retain its nucleophilicity. In a normal enzyme reaction, the hydrophobic environment is supposed to prevent hydration of the highly electronegative fluorine atom of the substrate and contribute to fluorine recognition by the enzyme. Basic residues probably play a role in counterbalancing the electronegativity of the substrate. These results demonstrate that catalysis-linked inactivation is useful for charac-terizing the active-site environment as well as for identifying the catalytic residue.
- 社団法人 日本生化学会の論文
著者
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Kurihara Tatsuo
Laboratory Of Microbial Biochemistry Institute For Chemical Research Kyoto University
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Esaki Nobuyoshi
Laboratory Of Microbial Biochemistry Institute For Chemical Research Kyoto University
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KAWASAKI Haruhiko
Department of Agricultural Chemistry, University of Osaka Prefecture
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Ichiyama Susumu
Laboratory Of Microbial Biochemistry Institute For Chemical Research Kyoto University
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Kawasaki Haruhiko
Department Of Agricultural Chemistry College Of Agriculture University Of Osaka Prefecture
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Galkin Andrey
Laboratory Of Microbial Biochemistry Institute For Chemical Research Kyoto University
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Miyagi Masaru
Biotechnology Research Laboratories Takara Shuzo Co.
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Tsunasawa Susumu
Biotechnology Research Laboratories Takara Shuzo Co. Ltd.
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