Specificity and kinetics of haloalkane dehalogenase.
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.
Schanstra
J P
JP
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Kingma
J
J
Janssen
D B
DB
D001665:Binding Sites; D017666:Deuterium Oxide; D004926:Escherichia coli / Q000201:enzymology; D015946:Ethylene Dibromide / Q000378:metabolism; D005025:Ethylene Dichlorides / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D005025:Ethylene Dichlorides; D011994:Recombinant Proteins; D015946:Ethylene Dibromide; C024565:ethylene dichloride; D006867:Hydrolases; C046137:haloalkane dehalogenase; D017666:Deuterium Oxide