Insights into the catalytic mechanism of a bacterial hydrolytic dehalogenase that degrades the fungicide chlorothalonil.

Chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile; TPN) 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 sp. (Chd), enabling determination of its metal-binding properties; pH dependence of the kinetic parameters , , and / ; and solvent isotope effects. We found that a single Zn(II) ion binds a Chd monomer with a of 0.17 μm, consistent with inductively coupled plasma 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 p of 5.4 ± 0.2, a p of 9.9 ± 0.1 (' = 24 ± 2 s), a p of 5.4 ± 0.3, and a p of 9.5 ± 0.1 ('/' = 220 ± 10 s mm). Proton inventory studies indicated that one proton is transferred in the rate-limiting step of the reaction at pD 7.0. Fits of UV-visible stopped-flow data suggested a three-step model and provided apparent rate constants for intermediate formation ( a ' of 35.2 ± 0.1 s) and product release ( a ' of 1.1 ± 0.2 s), 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.