Reaction Mechanism of L-2-Haloacid Dehalogenase of Pseudomonas sp. YL

L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalogenation of L-2-haloacids to produce the corresponding D-2-hydroxy acids. We have analyzed the reaction mechanism of the enzyme from Pseudomonas sp. YL and found that Asp is the active site nucleophile. When the multiple turnover enzyme reaction was carried out in H218O with L-2-chloropropionate as a substrate, lactate produced was labeled with 18O. However, when the single turnover enzyme reaction was carried out by use of a large excess of the enzyme, the product was not labeled. This suggests that an oxygen atom of the solvent water is first incorporated into the enzyme and then transferred to the product. After the multiple turnover reaction in H218O, the enzyme was digested with lysyl endopeptidase, and the molecular masses of the peptide fragments formed were measured by an ionspray mass spectrometer. Two 18O atoms were shown to be incorporated into a hexapeptide, Gly6-Lys. Tandem mass spectrometric analysis of this peptide revealed that Asp was labeled with two 18O atoms. Our previous site-directed mutagenesis experiment showed that the replacement of Asp led to a significant loss in the enzyme activity. These results indicate that Asp acts as a nucleophile on the α-carbon of the substrate leading to the formation of an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon atom.