Mechanism of the Reaction Catalyzed by dl-2-Haloacid Dehalogenase As Determined from Kinetic Isotope Effects

• Published in: Biochemistry (Easton) 2006-05, Vol.45 (19), p.6012-6017
• Main Authors: Papajak, Ewa, Kwiecień, Renata A, Rudziński, Juliusz, Sicińska, Daria, Kamiński, Rafał, Szatkowski, Łukasz, Kurihara, Tatsuo, Esaki, Nobuyoshi, Paneth, Piotr
• Format: Article
• Language: English
• Subjects: Hydrolases - chemistry; Hydrolases - metabolism; Kinetics; Models, Molecular; Spectrometry, Mass, Fast Atom Bombardment; Spectrophotometry, Ultraviolet
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0519553

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.