title: Replacement of tryptophan residues in haloalkane dehalogenase reduces halide binding and catalytic activity.
abstract: Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in short-chain haloalkanes. Two tryptophan residues of the enzyme (Trp125 and Trp175) form a halide-binding site in the active-site cavity, and were proposed to play a role in catalysis. The function of these residues was studied by replacing Trp125 with phenylalanine, glutamine or arginine and Trp175 by glutamine using site-directed mutagenesis. All mutants except Trp125-->Phe showed a more than 10-fold reduced kcat and much higher Km values with 1,2-dichloroethane and 1,2-dibromoethane than the wild-type enzyme. Fluorescence quenching experiments showed a decrease in the affinity of the mutant enzymes for halide ions. The 2H kinetic isotope effect observed with the wild-type enzyme in deuterium oxide was lost in the active mutants, except the Trp125-->Phe enzyme. The results indicate that both tryptophans are involved in stabilizing the transition state during the nucleophilic substitution reaction that causes carbon-halogen bond cleavage.
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
authors.forename: C
authors.initials: C
authors.lastname: Kennes
authors.forename: F
authors.initials: F
authors.lastname: Pries
authors.forename: G H
authors.initials: GH
authors.lastname: Krooshof
authors.forename: E
authors.initials: E
authors.lastname: Bokma
authors.forename: J
authors.initials: J
authors.lastname: Kingma
authors.forename: D B
authors.initials: DB
authors.lastname: Janssen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001965:Bromides / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D017666:Deuterium Oxide / Q000494:pharmacology; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D014364:Tryptophan
chemical_list: D001965:Bromides; D002712:Chlorides; D014364:Tryptophan; D006867:Hydrolases; C046137:haloalkane dehalogenase; D017666:Deuterium Oxide