Reaction of rat liver glutathione S-transferases and bacterial dichloromethane dehalogenase with dihalomethanes

Dichloromethane dehalogenase from Methylophilus sp. DM11 is a glutathione S-transferase homolog that is specifically active with dihalomethane substrates. This bacterial enzyme and rat liver glutathione S-transferases were purified to investigate their relative reactivity with CH₂Cl₂ and related substrates. Rat liver α class glutathione transferases were inactive and μ class enzymes showed low activity (7-23 nmol/min/mg of protein) with CH₂Cl₂. θ class glutathione transferase 5-5 from rat liver and Methylophilus sp. dichloromethane dehalogenase showed specific activities of ≥1 μmol/min/mg of protein. Apparent K(cat)/K(m) were determined to be 3.3 x 10⁴ and 6.0 x 10⁴ L M^-1 S^-1 for the two enzymes, respectively. Dideutereo-dichloromethane was processed to dideutereo-formaldehyde, consistent with a nucleophilic halide displacement mechanism. The possibility of a GSCH₂X reaction intermediate (GS, glutathione; X, halide) was probed using CH₂ClF to generate a more stable halomethylglutathione species (GSCH₂F). The reaction of CH₂ClF with dichloromethane dehalogenase produced a kinetically identifiable intermediate that decomposed to formaldehyde at a similar rate to synthetic HOCH₂CH₂SCH₂F. ¹⁹F-NMR revealed the transient formation of an intermediate identified as GSCH₂F by its chemical shift, its triplet resonance, and H-F coupling constant consistent with a fluoromethylthioether. Its decomposition was matched by a stoichiometric formation of fluoride. These studies indicated that the bacterial dichloromethane dehalogenase directs a nucleophilic attack of glutathione on CH₂Cl₂ to produce a halomethylthioether intermediate. This focuses attention on the mechanism used by θ class glutathione transferases to generate a halomethylthioether from relatively unreactive dihalomethanes.