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.
Highlights
MPGES2 has been believed to be one of the enzymes that catalyze Prostaglandin E2 (PGE2) synthesis, but recent studies have produced contradictory results
Heme Content in Recombinant microsomal prostaglandin E synthase type 2 (mPGES2) Increases along with an Increase in the Iron Concentration in the Culture Medium— The cloned E. coli cells were cultured in three different media containing different amounts of iron. (Minimal medium containing 5% LB medium had a nearly zero iron content, LB medium had an average iron content, and LB medium containing 0.25 mM Fe(NO3)3 had a high iron content.) SDS-PAGE of the cell extracts indicated that different iron contents did not alter the mPGES2
Because the crystal structure of mPGES2-hH indicates that the content of heme in each subunit is near 100% [6], the heme contents of mPGES2-lH and mPGES2-zH were calculated to be 78 and 8%, respectively
Summary
MPGES2 has been believed to be one of the enzymes that catalyze PGE2 synthesis, but recent studies have produced contradictory results. MPGES2 containing a GSH-heme complex catalyzes PGH2 degradation to (12S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid (HHT) and malondialdehyde (MDA) with a catalytic efficiency of 4.8 ϫ 105 MϪ1 sϪ1, but not PGE2 formation.
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