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 alpha class glutathione transferases were inactive and mu class enzymes showed low activity (7-23 nmol/min/mg of protein) with CH2Cl2. theta class glutathione transferase 5-5 from rat liver and Methylophilus sp. dichloromethane dehalogenase showed specific activities of > or = 1 mumol/min/mg of protein. Apparent Kcat/Km were determined to be 3.3 x 10(4) and 6.0 x 10(4) L M-1 S-1 for the two enzymes, respectively. Dideutero-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 theta class glutathione transferases to generate a halomethylthioeter from relatively unreactive dihalomethanes.
Blocki
F A
FA
Department of Biochemistry, University of Minnesota, Saint Paul 55108.
Logan
M S
MS
Baoli
C
C
Wackett
L P
LP
D000818:Animals; D017402:Chlorofluorocarbons / Q000378:metabolism; D005557:Formaldehyde / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008190:Lyases / Q000378:metabolism*; D008752:Methylene Chloride / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D017402:Chlorofluorocarbons; D005557:Formaldehyde; D008752:Methylene Chloride; C039310:chlorofluoromethane; D005982:Glutathione Transferase; D008190:Lyases; C045224:dichloromethane dehalogenase; D005978:Glutathione