Mechanism of the reaction catalyzed by DL-2-haloacid dehalogenase as determined from kinetic isotope effects
| Autor: | Rafał Kamiński, Łukasz Szatkowski, Tatsuo Kurihara, Juliusz Rudzinski, Renata A. Kwiecień, Nobuyoshi Esaki, Piotr Paneth, Ewa Papajak, Daria Sicinska |
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| Rok vydání: | 2006 |
| Předmět: |
Models
Molecular biology Chemistry Hydrolases Halogenation Spectrometry Mass Fast Atom Bombardment biology.organism_classification Biochemistry Medicinal chemistry Pseudomonas putida Catalysis Solvent Kinetics Deuterium Kinetic isotope effect Organic chemistry Spectrophotometry Ultraviolet Enantiomer Dehalogenase |
| Zdroj: | Biochemistry. 45(19) |
| ISSN: | 0006-2960 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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