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(Received for publication, January 18, 1996)
From the Department of Biochemistry, Groningen Biomolecular
Sciences and Biotechnology Institute, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands
Haloalkane dehalogenase converts halogenated
alkanes to their corresponding alcohols. The active site is buried
inside the protein and lined with hydrophobic residues. The reaction
proceeds via a covalent substrate-enzyme complex. This paper describes
a steady-state and pre-steady-state kinetic analysis of the conversion
of a number of substrates of the dehalogenase. The kinetic mechanism
for the ``natural'' substrate 1,2-dichloroethane and for the
brominated analog and nematocide 1,2-dibromoethane are given. In
general, brominated substrates had a lower Km, but
a similar kcat than the chlorinated analogs.
The rate of C-Br bond cleavage was higher than the rate of C-Cl bond
cleavage, which is in agreement with the leaving group abilities of
these halogens. The lower Km for brominated
compounds therefore originates both from the higher rate of C-Br bond
cleavage and from a lower Ks for
bromo-compounds. However, the rate-determining step in the conversion
(kcat) of 1,2-dibromoethane and
1,2-dichloroethane was found to be release of the charged halide ion
out of the active site cavity, explaining the different
Km but similar kcat values
for these compounds. The study provides a basis for the analysis of
rate-determining steps in the hydrolysis of various environmentally
important substrates.
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