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 α-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.