Abstract:
:Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols and halides. The reaction mechanism involves the formation of a covalent alkyl-enzyme complex which is hydrolyzed by water. The active site is a hydrophobic cavity buried between the main domain and the cap domain of the enzyme. The enzyme has a broad substrate specificity, but the kcat values of the enzyme for the best substrates 1,2-dichloroethane and 1,2-dibromoethane are rather low (3 and 3.5 s-1, respectively). Stopped-flow fluorescence experiments with substrate under single-turnover conditions indicated that halide release could limit the overall kcat. Furthermore, at 5mM 1,2-dibromoethane the observed rate of substrate binding to free enzyme was faster than 700 s-1 (within the dead time of the stopped-flow instrument) whereas displacement of halide by 5mM 1,2-dibromoethane occurred at a rate of only 8 s-1. The binding of bromide and chloride to free enzyme was also studied using stopped-flow fluorescence, and the dependence of kobs on the halide concentration suggested that there were two parallel routes for halide binding. One route, in which a slow enzyme isomerization is followed by rapid halide binding, was predominant at low halide concentrations. The other route involves rapid binding into an initial collision complex followed by a slow enzyme isomerization step and prevailed at higher halide concentrations. The overall rate of halide release was low and limited by a slow enzyme isomerization preceding actual release (9 and 14.5 s-1 for bromide and chloride, respectively). We propose that this slow isomerization is a conformational change in the cap domain that is necessary to allow water to enter and solvate the halide ion. A solvent kinetic isotope effect of 2H2O was found both on kcat and on the rate of halide release. 2H2O mainly affected the rate of the conformational change, which is in agreement with this step being rate-limiting and the overall stabilizing effect of 2H2O on the conformation of proteins.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Schanstra JP,Janssen DBdoi
10.1021/bi952904gsubject
Has Abstractpub_date
1996-05-07 00:00:00pages
5624-32issue
18eissn
0006-2960issn
1520-4995pii
bi952904gjournal_volume
35pub_type
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