title: Reaction mechanism of fluoroacetate dehalogenase from Moraxella sp. B.
abstract: 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., Koca, 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 alpha-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.
authors.affiliation: Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: J Q
authors.initials: JQ
authors.lastname: Liu
authors.forename: T
authors.initials: T
authors.lastname: Kurihara
authors.forename: S
authors.initials: S
authors.lastname: Ichiyama
authors.forename: M
authors.initials: M
authors.lastname: Miyagi
authors.forename: S
authors.initials: S
authors.lastname: Tsunasawa
authors.forename: H
authors.initials: H
authors.lastname: Kawasaki
authors.forename: K
authors.initials: K
authors.lastname: Soda
authors.forename: N
authors.initials: N
authors.lastname: Esaki
mesh_terms: D000085:Acetates / Q000378:metabolism; D000478:Alkylation; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics; D004952:Esters / Q000378:metabolism; D005463:Fluoroacetates / Q000378:metabolism*; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009016:Moraxella / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010103:Oxygen Isotopes; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D000085:Acetates; D004952:Esters; D005463:Fluoroacetates; D010103:Oxygen Isotopes; D011994:Recombinant Proteins; C006972:chloroacetic acid; D006867:Hydrolases; C023098:haloacetate dehalogenase; C046137:haloalkane dehalogenase