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

Abstract

Hematopoietic prostaglandin (PG) D synthase (PGDS) is the first identified vertebrate ortholog in the Sigma class of the glutathione S-transferase (GST) family and catalyzes both isomerization of PGH2 to PGD2 and conjugation of glutathione to 1-chloro-2,4-dinitrobenzene. We introduced site-directed mutations of Tyr8, Arg14, Trp104, Lys112, Tyr152, Cys156, Lys198, and Leu199, which are presumed to participate in catalysis or PGH2 substrafe binding based on the crystallographic structure. Mutants were analyzed in terms of structure, GST and PGDS activities, and activation of the glutathione thiol group. Of all the mutants, only Y8F, W104I, K112E, and L199F showed minor but substantial differences in their far-UV circular dichroism spectra from the wild-type enzyme. Y8F, R14K/E, and W104I were completely inactive. C156L/Y selectively lost only PGDS activity. K112E reduced GST activity slightly and PGDS activity markedly, whereas K198E caused a selective decrease in PGDS activity and K(m) for glutathione and PGH2 in the PGDS reaction. No significant changes were observed in the catalytic activities of Y152F and L199F, although their K(m) for glutathione was increased. Using 5,5'-dithiobis(2-nitrobenzoic acid) as an SH-selective agent, we found that only Y8F and R14E/K did not accelerate the reactivity of the glutathione thiol group under the low reactivity condition of pH 5.0. These results indicate that Lys112, Cys156, and Lys198 are involved in the binding of PGH2; Trp104 is critical for structural integrity of the catalytic center for GST and PGDS activities; and Tyr8 and Arg14 are essential for activation of the thiol group of glutathione.