Chlorothalonil (TPN; 2,4,5,6-tetrachloroisophtalonitrile) is one of the most commonly used fungicides in the United States. Given TPN's widespread use, general toxicity, and potential carcinogenicity, its biodegradation has garnered significant attention. Here, we developed a direct spectrophotometric assay for the Zn(II)-dependent, chlorothalonil-hydrolyzing dehalogenase from Pseudomonas sp. CTN-3 (Chd), enabling determination of its metal-binding properties; pH dependence of the kinetic parameters k cat, K m, and k cat/K m; and solvent isotope effects. We found that a single Zn(II) ion binds a Chd monomer with a K d of 0.17 μM, consistent with inductively coupled plasma (ICP) MS data for the as-isolated Ch... More
Chlorothalonil (TPN; 2,4,5,6-tetrachloroisophtalonitrile) is one of the most commonly used fungicides in the United States. Given TPN's widespread use, general toxicity, and potential carcinogenicity, its biodegradation has garnered significant attention. Here, we developed a direct spectrophotometric assay for the Zn(II)-dependent, chlorothalonil-hydrolyzing dehalogenase from Pseudomonas sp. CTN-3 (Chd), enabling determination of its metal-binding properties; pH dependence of the kinetic parameters k cat, K m, and k cat/K m; and solvent isotope effects. We found that a single Zn(II) ion binds a Chd monomer with a K d of 0.17 μM, consistent with inductively coupled plasma (ICP) MS data for the as-isolated Chd dimer. We observed that Chd was maximally active toward chlorothalonil in the pH range 7.0-9.0, and fits of these data yielded a pK ES1 of 5.4 ± 0.2, a pK ES2 of 9.9 ± 0.1 (k'cat = 24 ± 2 s-1), a pK E1 of 5.4 ± 0.3, and a pK E2 of 9.5 ± 0.1 (k'cat/K'm = 220 ± 10 s-1 mM-1). Proton inventory studies indicated that one proton is transferred in the rate-limiting step of the reaction at pD 7.0. Fits of UV-Vis stopped-flow data suggested a three-step model and provided apparent rate constants for intermediate formation, i.e. a ᵅ8;2' of 35.2 ± 0.1 s-1, and product release, i.e a ᵅ8;3' of 1.1 ± 0.2 s-1, indicating that product release is the slow-step in catalysis. On the basis of these results, along with those previously reported, we propose a mechanism for Chd catalysis.
