title: Trimeric microsomal glutathione transferase 2 displays one third of the sites reactivity.
abstract: Human microsomal glutathione transferase 2 (MGST2) is a trimeric integral membrane protein that belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family. The mammalian MAPEG family consists of six members where four have been structurally determined. MGST2 activates glutathione to form a thiolate that is crucial for GSH peroxidase activity and GSH conjugation reactions with electrophilic substrates, such as 1-chloro-2,4-dinitrobenzene (CDNB). Several studies have shown that MGST2 is able to catalyze a GSH conjugation reaction with the epoxide LTA4 forming the pro-inflammatory LTC4. Unlike its closest homologue leukotriene C4 synthase (LTC4S), MGST2 appears to activate its substrate GSH using only one of the three potential active sites [Ahmad S, et al. (2013) Biochemistry. 52, 1755-1764]. In order to demonstrate and detail the mechanism of one-third of the sites reactivity of MGST2, we have determined the enzyme oligomeric state, by Blue native PAGE and Differential Scanning Calorimetry, as well as the stoichiometry of substrate and substrate analog inhibitor binding to MGST2, using equilibrium dialysis and Isothermal Titration Calorimetry, respectively. Global simulations were used to fit kinetic data to determine the catalytic mechanism of MGST2 with GSH and CDNB (1-chloro-2,4-dinitrobenzene) as substrates. The best fit was observed with 1/3 of the sites catalysis as compared with a simulation where all three sites were active. In contrast to LTC4S, MGST2 displays a 1/3 the sites reactivity, a mechanism shared with the more distant family member MGST1 and recently suggested also for microsomal prostaglandin E synthase-1.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Scheeles väg 2, Karolinska Institutet, 17177 Stockholm, Sweden. Electronic address: hasan.ahmad@ki.se.
authors.forename: Shabbir
authors.initials: S
authors.lastname: Ahmad
authors.affiliation: Department of Medical Biochemistry and Biophysics, Scheeles väg 2, Karolinska Institutet, 17177 Stockholm, Sweden. Electronic address: Madhuranayaki.thulasingam@ki.se.
authors.forename: Madhuranayaki
authors.initials: M
authors.lastname: Thulasingam
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden. Electronic address: Isolde.palombo@ki.se.
authors.forename: Isolde
authors.initials: I
authors.lastname: Palombo
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden. Electronic address: Dan.Daley@dbb.su.se.
authors.forename: Daniel O
authors.initials: DO
authors.lastname: Daley
authors.affiliation: Department of Molecular Biosciences, University of Texas, Austin, TX, USA. Electronic address: kajohnson@mail.utexas.edu.
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Johnson
authors.affiliation: Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden. Electronic address: Ralf.Morgenstern@ki.se.
authors.forename: Ralf
authors.initials: R
authors.lastname: Morgenstern
authors.affiliation: Department of Medical Biochemistry and Biophysics, Scheeles väg 2, Karolinska Institutet, 17177 Stockholm, Sweden. Electronic address: Jesper.Haeggstrom@ki.se.
authors.forename: Jesper Z
authors.initials: JZ
authors.lastname: Haeggström
authors.affiliation: Department of Medical Biochemistry and Biophysics, Scheeles väg 2, Karolinska Institutet, 17177 Stockholm, Sweden. Electronic address: Agnes.Rinaldo-Matthis@ki.se.
authors.forename: Agnes
authors.initials: A
authors.lastname: Rinaldo-Matthis
mesh_terms: D000595:Amino Acid Sequence; D002151:Calorimetry; D020134:Catalytic Domain; D004137:Dinitrochlorobenzene / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D004137:Dinitrochlorobenzene; D011994:Recombinant Proteins; C122107:microsomal glutathione S-transferase-I; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase; D005978:Glutathione
keywords: Global data fitting; Leukotriene C(4) synthase; MAPEG protein family; Microsomal glutathione transferase 2; Pre-steady state kinetics; Thiolate anion