Reaction mechanism of fluoroacetate dehalogenase from Moraxella sp. B

J Biol Chem. 1998 Nov 20;273(47):30897-902. doi: 10.1074/jbc.273.47.30897.

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.

Publication types

  • Comparative Study

MeSH terms

  • Acetates / metabolism
  • Alkylation
  • Amino Acid Sequence
  • Catalytic Domain
  • Escherichia coli / genetics
  • Esters / metabolism
  • Fluoroacetates / metabolism*
  • Hydrolases / genetics
  • Hydrolases / metabolism*
  • Hydrolysis
  • Mass Spectrometry
  • Molecular Sequence Data
  • Moraxella / enzymology*
  • Moraxella / genetics
  • Mutagenesis, Site-Directed
  • Oxygen Isotopes
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid

Substances

  • Acetates
  • Esters
  • Fluoroacetates
  • Oxygen Isotopes
  • Recombinant Proteins
  • chloroacetic acid
  • Hydrolases
  • haloacetate dehalogenase
  • haloalkane dehalogenase