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 (GST) family and catalyzes both isomerization of PGH(2) to PGD(2) 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(198), and Leu(199), which are presumed to participate in catalysis or PGH(2) substrate 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 PGH(2) 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 Lys(112), Cys(156), and Lys(198) are involved in the binding of PGH(2); Trp(104) is critical for structural integrity of the catalytic center for GST and PGDS activities; and Tyr(8) and Arg(14) are essential for activation of the thiol group of glutathione.
Pinzar
E
E
Department of Molecular Behavioral Biology and Core Research for Evolutional Science and Technology, Japan Science Technology Corporation, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan.
Miyano
M
M
Kanaoka
Y
Y
Urade
Y
Y
Hayaishi
O
O
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004136:Dinitrobenzenes / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000201:enzymology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D019746:Intramolecular Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D054834:Lipocalins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D014466:Ultraviolet Rays
D000596:Amino Acids; D017931:DNA Primers; D004136:Dinitrobenzenes; D054834:Lipocalins; D011994:Recombinant Proteins; D014443:Tyrosine; D001120:Arginine; D005982:Glutathione Transferase; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D005978:Glutathione; C010936:2,4-dinitrothiocyanatobenzene