Mechanism of the reaction catalyzed by DL-2-haloacid dehalogenase as determined from kinetic isotope effects

DL- 2- Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of (D)- 2- haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on ( R)-(+)- 2- haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the alpha- carbon of the 2- haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 +/- 0.0001 for ( S)-(-)- 2- chloropropionate and 1.0082 +/- 0.0005 for the ( R)-(+)- isomer. Together with solvent deuterium isotope effects on V-max/ K-M, 0.78 +/- 0.09 for ( S)-(-)- 2- chloropropionate and 0.90 +/- 0.13 for the ( R)-(+)- isomer, these values indicate that in the case of the ( R)-(+)- reactant another step preceding the dehalogenation is partly rate- limiting. Under the V-max conditions, the corresponding solvent deuterium isotope effects are 1.48 +/- 0.10 and 0.87 +/- 0.27, respectively. These results indicate that the overall reaction rates are controlled by different steps in the catalysis of ( S)-(-)- and ( R)-(+)- reactants.