Haloalkane Dehalogenases: Steady-State Kinetics and Halide Inhibition

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Abstract
The substrate specificities and product inhibition patterns of haloalkane dehalogenases from Xanthobacterautotrophicus GJ10 (XaDHL) and Rhodococcusrhodochrous (RrDHL) have been compared using a pH-indicator dye assay. In contrast to XaDHL, RrDHL is efficient toward secondary alkyl 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-, Kii = 19 ± 0.91; Br-, Kii = 2.5 ± 0.19 mM; I-, Kii = 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 Kii 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/Kii showed simple titration behavior that fit to a pKa of approximately 7.5. The kcat was maximal at pH 8.2 and decreased at lower pH. A titration of kcat versus pH also fits to a pKa of approximately 7.5. Taken together, these data suggest that chloride binding and kcat 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.