Structural basis of hematopoietic prostaglandin D synthase activity elucidated by site-directed mutagenesis

Hematopoietic prostaglandin (PG) D synthase (PGDS) is the first identified vertebrate ortholog in the Sigma class of the glutathione S-transferase (GS T) family and catalyzes both isomerization of PGH, to PGD, and conjugation of glutathione to 1-chloro-2,4-dinitrobenzene. We introduced site directed mutations of Tyr(8), Arg(14), Trp(104), Lys(112), Tyr(152), Cys(156), Lys(1 98), and Leu(199), which are presumed to participate in catalysis or PGH(2) substrate binding based on the crystallographic structure. Mutants were an alyzed in terms of structure, GST and PGDS activities, and activation of th e glutathione thiol group. Of all the mutants, only Y8F, W104I, K112E, and L199F showed minor but substantial differences in their far-UV circular dic hrcoism spectra from the wild-type enzyme, Y8F, R14K/E, and W104I were comp letely inactive. C156L/Y selectively lost only PGDS activity. K112E reduced GST activity slightly and PGDS activity markedly, whereas K198E caused a s elective decrease in PODS activity and K-m for glutathione and PGH(2) in th e PODS reaction. No significant changes were observed in the catalytic acti vities of Y152F and L199F, although their K-m for glutathione was increased . Using 5,5'-dithiobis(2-nitrobenzoic acid) as an SE-selective agent, we fo und that only Y8F and R14E/K did not accelerate the reactivity of the gluta thione thiol group under the low reactivity condition of pH 5.0. These resu lts indicate that Lys(112), Cys(156) and Lys(198) are involved in the bindi ng of PGH,; Trp(104) is critical for structural integrity of the catalytic center for GST and PODS activities; and Tyr(8) and Arg(14), essential for a ctivation of the thiol group of glutathione.