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J Biol Chem, Vol. 273, Issue 47, 30897-30902, November 20, 1998

Reaction Mechanism of Fluoroacetate Dehalogenase from Moraxella sp. B

Ji-Quan Liu, Tatsuo Kurihara, Susumu Ichiyama, Masaru Miyagi^§, Susumu Tsunasawa^§, Haruhiko Kawasaki^¶, Kenji Soda, and Nobuyoshi Esaki

From the  Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan, ^§ Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Kusatsu, Shiga 525-0055, Japan, and the ^¶ Department of Agricultural Chemistry, University of Osaka Prefecture, Sakai, Osaka 599-8531, Japan

Fluoroacetate dehalogenase (EC 3.8.1.3) catalyzes the dehalogenation of fluoroacetate and other haloacetates. The amino acid sequence of fluoroacetate dehalogenase from Moraxella sp. B is similar to that of haloalkane dehalogenase (EC 3.8.1.5) from Xanthobacter autotrophicus GJ10 in the regions around Asp-105 and His-272, which correspond to the active site nucleophile Asp-124 and the base catalyst His-289 of the haloalkane dehalogenase, respectively (Krooshof, G. H., Kwant, E. M., Damborský, J., Koa, J., and Janssen, D. B. (1997) Biochemistry 36, 9571-9580). After multiple turnovers of the fluoroacetate dehalogenase reaction in H₂¹⁸O, the enzyme was digested with trypsin, and the molecular masses of the peptide fragments formed were measured by ion-spray mass spectrometry. Two ¹⁸O atoms were shown to be incorporated into the octapeptide, Phe-99-Arg-106. Tandem mass spectrometric analysis of this peptide revealed that Asp-105 was labeled with two ¹⁸O atoms. These results indicate that Asp-105 acts as a nucleophile to attack the -carbon of the substrate, leading to the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule on the carbonyl carbon atom. A His-272  Asn mutant (H272N) showed no activity with either fluoroacetate or chloroacetate. However, ion-spray mass spectrometry revealed that the H272N mutant enzyme was covalently alkylated with the substrate. The reaction of the H272N mutant enzyme with [¹⁴C]chloroacetate also showed the incorporation of radioactivity into the enzyme. These results suggest that His-272 probably acts as a base catalyst for the hydrolysis of the covalent ester intermediate.

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