title: Microsomal prostaglandin E synthase type 2 (mPGES2) is a glutathione-dependent heme protein, and dithiothreitol dissociates the bound heme to produce active prostaglandin E2 synthase in vitro.
abstract: An x-ray study indicated that microsomal prostaglandin E synthase type 2 (mPGES2) is a heme-bound protein and catalyzes prostaglandin (PG) H2 degradation, but not PGE2 formation (Yamada, T., and Takusagawa, F. (2007) Biochemistry 46, 8414-8424). In response to the x-ray study, Watanabe et al. claimed that mPGES2 is a heme-free protein and that both the heme-free and heme-bound proteins have PGE2 synthesis activity in the presence of dithiothreitol (Watanabe, K., Ito, S., and Yamamoto, S. (2008) Biochem. Biophys. Res. Commun. 367, 782-786). To resolve the contradictory results, the heme-binding scheme of mPGES2 was further characterized in vivo and in vitro by absorption and fluorescence spectroscopies. A substantial amount of heme-bound mPGES2 was detected in cell extracts. The heme content in mPGES2 was increased along with an increase in Fe(3+) in the culture medium. Heme-free mPGES2 was converted to the heme-bound form by mixing it with pig liver extract, indicating that mPGES2 is capable of forming a complex with heme in mammalian cells. Heme binds to mPGES2 only in the presence of glutathione. The newly determined heme dissociation constant (2.9 nM) supports strongly that mPGES2 is a heme-bound protein in vivo. The bound heme was not dissociated by oxidation by H2O2 or reduction by glutathione or 2-mercaptoethanol. However, reduction by dithiothreitol (an artificial reducing compound) induced the bound heme to dissociate from mPGES2 and released heme-free mPGES2, which exhibited PGE2 synthesis activity in vitro. Imidazole bound to mPGES2 by stacking on the bound heme and inhibited heme oxidation by H2O2 and reduction by dithiothreitol.
authors: Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045-7534. Electronic address: xraymain@ku.edu.
authors: Fusao
authors: F
authors: Takusagawa
mesh_terms: D000818:Animals; D002384:Catalysis; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006418:Heme / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D019746:Intramolecular Oxidoreductases / Q000378:metabolism*; D008252:Macaca fascicularis; D008623:Mercaptoethanol / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology*; D008958:Models, Molecular; D000071876:Prostaglandin-E Synthases; D011485:Protein Binding; D013552:Swine
chemical_list: D006418:Heme; D008623:Mercaptoethanol; D006861:Hydrogen Peroxide; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases; D005978:Glutathione; D004229:Dithiothreitol