Haloalkane Dehalogenases:  Steady-State Kinetics and Halide Inhibition

Abstract

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 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.

Steady-State and Pre-Steady-State Kinetic Analysis of Halopropane Conversion by a Rhodococcus Haloalkane Dehalogenase

“…Both chloro-and bromopropanes are substrates for DhaA (Table 1). In agreement with previous results, bromopropanes are better substrates with turnover numbers (k cat ) that are higher than those of chlorinated analogues (25,26). In terms of catalytic efficiency (k cat /K M ), 1,3-dibromopropane was the best substrate.…”

“…The latter dehalogenase exhibits the highest activity on smaller compounds such as its natural substrate 1,2-dichloroethane and the brominated analogue and nematocide 1,2-dibromoethane (20). Interestingly, DhaA converted 1,2-dibromoethane at a steady-state rate that is 4-5-fold higher than that found with DhlA, even though 1,2-dichloroethane is not a substrate for DhaA (20,25,26).…”

“…This suggests that bromide exerts a noncompetitive type of inhibition, most likely mixed, on DhaA. Surprisingly, Schindler et al (25), using an assay dependent on pH changes, did not find halide inhibition of DhaA at concentrations up to 80 mM. Comparing the maximum turnover rates of DhaA with halopropanes using H 2 O or 2 H 2 O as a solvent provides information about the position of the slowest step in the catalytic cycle.…”

Kinetic Mechanism and Enantioselectivity of Halohydrin Dehalogenase from Agrobacterium radiobacter

“…Thus, halide binding and release are separated from free enzyme by a conformational change (30). This explains that with DhlA halide is a noncompetitive inhibitor, even though it is the last product that is released, an explanation that has been overlooked by others (31). However, this cannot be the case for HheC since the singleturnover experiments revealed that the second product is released immediately after the first, indicating that the first product release step is rate-limiting.…”

Biotransformation of halogenated compounds by lyophilized cells of Rhodococcus erythropolis in a continuous solid–gas biofilter

“…Indeed, in aqueous solutions, it has been shown that the halides are uncompetitive inhibitors of haloalkane dehalogenase from Rhodococcus sp., and the K i for chloride and bromide was pH-dependent [28]. Consequently, in the reaction of dehalogenation by the haloalkane dehalogenase, the rate-limiting step is the release of the halogen ion from the active site.…”