REPLACEMENT OF TRYPTOPHAN RESIDUES IN HALOALKANE DEHALOGENASE REDUCESHALIDE BINDING AND CATALYTIC ACTIVITY

Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-ha logen bonds in short-chain haloalkanes. Two tryptophan residues of the enzyme (Trp125 and Trp175) form a halide-binding site in the active-s ite cavity, and were proposed to play a role in catalysis. The functio n of these residues was studied by replacing Trp125 with phenylalanine , glutamine or arginine and Trp175 by glutamine using site-directed mu tagenesis. All mutants except Trp125-->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 ex periments showed a decrease in the affinity of the mutant enzymes for halide ions. The H-2 kinetic isotope effect observed with the wild-typ e enzyme in deuterium oxide was lost in the active mutants, except the Trp125-->Phe enzyme. The results indicate that both tryptophans are i nvolved in stabilizing the transition state during the nucleophilic su bstitution reaction that causes carbon-halogen bond cleavage.