Reaction Mechanism of DL-2-Haloacid Dehalogenase from Pseudomonas sp. 113: Hydrolytic Dehalogenation Not Involving Enzyme-Substrate Ester Intermediate (MOLECULAR BIOFUNCTION-Molecular Microbial Science)

Abstract

DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 (DL-DEX 113) catalyzes the hydrolytic dehalogenation of D- and L-2-haloalkanoic acids, producing the corresponding L- and D-2- hydroxyalkanoic acids, respectively. L-2-Haloacid dehalogenase, haloalkane dehalogenase, and 4- chlorobenzoyl-CoA dehalogenase, which catalyze the hydrolysis of various organohalogen compounds, have an active site carboxylate group that attacks the substrate carbon atom bound to the halogen atom, leading to the formation of an ester intermediate. This is subsequently hydrolyzed, resulting in the incorporation of an oxygen atom of the solvent water molecule into the carboxylate group of the enzyme. In the present study, we analyzed the reaction mechanism of DL-DEX 113. When a single turnover reaction of DL-DEX 113 was carried out with a large excess of the enzyme in H2 18O with either D- or L-2-chloropropionate, the major product was found to be 18O-labeled lactate. After a multiple turnover reaction in H2 18O, the enzyme was digested with proteases, and the molecular masses of the peptide fragments were measured. No peptide fragments contained 18O. These results indicate that the H2 18O of the solvent directly attacks the α-carbon of 2-haloalkanoic acid to displace the halogen atom. This is the first example of an enzymatic hydrolytic dehalogenation that proceeds without formation of an ester intermediate.