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 Trp1l25→Phe showed a more than 10‐fold reduced K cat and much higher K m 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 2 H kinetic isotope effect observed with the wild‐type enzyme in deuterium oxide was lost in the active mutants, except the Trp 125 →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.