Haloalkane dehalogenases: Steady-state kinetics and halide inhibition

The substrate specificities and product inhibition patterns of haloalkane dehalogenases from Xanthobacter autotrophicus GJ10 (XaDHL) and Rhodococcus rhodochrous (RrDHL) have been compared using a pH-indicator dye assay. In contrast to XaDHL, RrDHL is efficient toward secondary allyl halides. Using steady-state kinetics, we have shown that halides are uncompetitive inhibitors of XaDHL with 1,2-dichloroethane as the varied substrate at pH 8.2 (Cl-, K-ii = 19 +/- 0.91; Br-, K-ii = 2.5 +/- 0.19 mM; I-, K-ii = 4.1 +/- 0.43 mM). Because they are uncompetitive with the substrate, halide ions do not bind to the free form of the enzyme; therefore, halide ions cannot be the last product released from the enzyme. The K-ii for chloride was pH dependent and decreased more than 20-fold from 61 mM at pH 8.9 to 2.9 mM at pH 6.5. The pH dependence of 1/K-ii showed simple titration behavior that fit to a pK(a) of approximately 7.5. The k(cat) was maximal at pH 8.2 and decreased at lower pH. A titration of k(cat) versus pH also fits to a pK(a) of approximately 7.5. Taken together, these data suggest that chloride binding and k(cat) are affected by the same ionizable group, likely the imidazole of a histidyl residue. In contrast, halides do not inhibit RrDHL. The Rhodococcus enzyme does not contain a tryptophan corresponding to W175 of XaDHL, which has been implicated in halide ion binding. The site-directed mutants W175F and W175Y of XaDHL were prepared and tested for halide ion inhibition. Halides do not inhibit either W175F or W175Y XaDHL.