4550
J. Am. Chem. Soc. 2001, 123, 4550-4555
Chlorine Kinetic Isotope Effects on the Haloalkane Dehalogenase
Reaction
†
†
‡
‡
Andrzej Lewandowicz, Juliusz Rudzi n´ ski, Lisa Tronstad, Mikael Widersten,
§
§
,†
Per Ryberg, Olle Matsson, and Piotr Paneth*
Department of Chemistry, Technical UniVersity of Lodz, Zeromskiego 116, 90-924 Lodz, Poland,
Department of Biochemistry, Biomedical Center, Uppsala UniVersity, S-751 23 Uppsala, Sweden,
Department of Chemistry, Uppsala UniVersity, S-751 21 Uppsala, Sweden
ReceiVed September 26, 2000. ReVised Manuscript ReceiVed March 7, 2001
Abstract: We have found chlorine kinetic isotope effects on the dehalogenation catalyzed by haloalkane
dehalogenase from Xanthobacter autotrophicus GJ10 to be 1.0045 ( 0.0004 for 1,2-dichloroethane and 1.0066
(
0.0004 for 1-chlorobutane. The latter isotope effect approaches the intrinsic chlorine kinetic isotope effect
for the dehalogenation step. The intrinsic isotope effect has been modeled using semiempirical and DFT theory
levels using the ONIOM QM/QM scheme. Our results indicate that the dehalogenation step is reversible; the
overall irreversibility of the enzyme-catalyzed reaction is brought about by a step following the dehalogenation.
Introduction
In recent years, mechanisms of action of various dehaloge-
1
nases have become a subject of intensive study as a result of
their potential use in bioremedation,2 because halogenated
organic compounds constitute the largest group of pollutants.
Most of these enzymes react through formation of a covalently
bonded intermediate, which is subsequently hydrolyzed to
product,1p although a direct displacement by a water molecule
1
c
activated by an enzyme base has recently been reported.
The reaction that is catalyzed by haloalkane dehalogenase
Figure 1. Chemical steps catalyzed by haloalkane dehalogenase.
from Xanthobacter autotrophicus GJ10 has been studied
3
extensively. This enzyme catalyzes hydrolysis of the C-Cl
Figure 1) is an SN2 reaction, in which the chlorine atom is
displaced by one of the carboxylic oxygens of the aspartate
bond of a carcinogenic pollutant, 1,2-dichloroethane [(ClCH2)2].
It was concluded that the first chemical step (top reaction in
(Asp124) residue. This dehalogenation step, which leads to the
†
Technical University of Lodz.
Department of Biochemistry, Biomedical Center, Uppsala University.
Department of Chemistry, Uppsala University.
‡
enzyme-bounded intermediate, is believed to be irreversible. In
the following reactions, abbreviated in Figure 1 to a single step,
this intermediate is hydrolyzed by the nearby water molecule
to the product. The chloride ion is hydrogen-bonded to two
§
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0.1021/ja003503d CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/14/2001