title: Catalytic mechanism of dichloromethane dehalogenase from Methylophilus sp. strain DM11.
abstract: The glutathione (GSH)-dependent dichloromethane dehalogenase from Methylophilus sp. strain DM11 catalyzes the dechlorination of CH(2)Cl(2) to formaldehyde via a highly reactive, genotoxic intermediate, S-(chloromethyl)glutathione (GS-CH(2)Cl). The catalytic mechanism of the enzyme toward a series of dihalomethane and monohaloethane substrates suggests that the initial addition of GSH to the alkylhalides is fast and that the rate-limiting step in turnover is the release of either the peptide product or formaldehyde. With the exception of CH(2)ClF, which forms a relatively stable GS-CH(2)F intermediate, the turnover numbers for a series of dihalomethanes fall in a very narrow range (1-3 s(-1)). The pre-steady-state kinetics of the DM11-catalyzed addition of GSH to CH(3)CH(2)Br exhibits a burst of S-(ethyl)-glutathione (k(b) = 96 +/- 56 s(-1)) followed by a steady state with k(cat) = 0.13 +/- 0.01 s(-1). The turnover numbers for CH(3)CH(2)Cl, CH(3)CH(2)Br, and CH(3)CH(2)I are identical, indicating a common rate-limiting step. The turnover numbers of the enzyme with CH(3)CH(2)Br and CH(3)CH(2)I are dependent on viscosity and are very close to the measured off-rate of GSEt. The turnover number with CH(2)I(2) is also dependent on viscosity, suggesting that a diffusive step is rate-limiting with dihaloalkanes as well. The rate constants for solvolysis of CH(3)SCH(2)Cl, a model for GS-CH(2)Cl, range between 1 s(-1) (1:1 dioxane/water) and 64 s(-1) (1:10 dioxane/water). Solvolysis of the S-(halomethyl)glutathione intermediates may also occur in the active site of the enzyme preventing the release of the genotoxic species. Together, the results indicate that dissociation of the GS-CH(2)X or GS-CH(2)OH intermediates from the enzyme may be a relatively rare event.
authors.affiliation: Department of Biochemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: Nina V
authors.initials: NV
authors.lastname: Stourman
authors.forename: James H
authors.initials: JH
authors.lastname: Rose
authors.forename: Stephane
authors.initials: S
authors.lastname: Vuilleumier
authors.forename: Richard N
authors.initials: RN
authors.lastname: Armstrong
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004058:Diffusion; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005557:Formaldehyde / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007454:Iodides / Q000737:chemistry; D007700:Kinetics; D008190:Lyases / Q000737:chemistry*; D020629:Methylophilus / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D011485:Protein Binding; D016958:Pseudomonas putida / Q000378:metabolism; D013997:Time Factors; D014783:Viscosity
chemical_list: D007454:Iodides; D005557:Formaldehyde; D008190:Lyases; C045224:dichloromethane dehalogenase; D005978:Glutathione