Crystal structure and possible catalytic mechanism of microsomal prostaglandin E synthase type 2 (mPGES-2)

Prostaglandin (PG) H(2) (PGH(2)), formed from arachidonic acid, is an unstable intermediate and is converted efficiently into more stable arachidonate metabolites (PGD(2), PGE(2), and PGF(2)) by the action of three groups of enzymes. Prostaglandin E synthase catalyzes an isomerization reaction, PGH(2) to PGE(2). Microsomal prostaglandin E synthase type-2 (mPGES-2) has been crystallized with an anti-inflammatory drug indomethacin (IMN), and the complex structure has been determined at 2.6A resolution. mPGES-2 forms a dimer and is attached to lipid membrane by anchoring the N-terminal section. Two hydrophobic pockets connected to form a V shape are located in the bottom of a large cavity. IMN binds deeply in the cavity by placing the OMe-indole and chlorophenyl moieties into the V-shaped pockets, respectively, and the carboxyl group interacts with S(gamma) of C110 by forming a H-bond. A characteristic H-bond chain formation (N-H...S(gamma)-H...S(gamma)...H-N) is seen through Y107-C113-C110-F112, which apparently decreases the pK(a) of S(gamma) of C110. The geometry suggests that the S(gamma) of C110 is most likely the catalytic site of mPGES-2. A search of the RCSB Protein Data Bank suggests that IMN can fit into the PGH(2) binding site in various proteins. On the basis of the crystal structure and mutation data, a PGH(2)-bound model structure was built. PGH(2) fits well into the IMN binding site by placing the alpha and omega-chains in the V-shaped pockets, and the endoperoxide moiety interacts with S(gamma) of C110. A possible catalytic mechanism is proposed on the basis of the crystal and model structures, and an alternative catalytic mechanism is described. The fold of mPGES-2 is quite similar to those of GSH-dependent hematopoietic prostaglandin D synthase, except for the two large loop sections.     

Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages.

We cloned the cDNA for mouse microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and expressed the recombinant enzyme in Escherichia coli. The membrane fraction containing recombinant mPGES-1 catalyzed the isomerization of PGH2 to PGE2 in the presence of GSH with K(m) values of 130 microM for PGH2 and 37 microM for GSH, a turnover number of 600 min(-1), and a k(cat)/K(m) ratio of 4.6 min(-1) microM(-1). Recombinant mPGES-1 was purified and used to generate a polyclonal antibody highly specific for mPGES-1. The antibody showed a single band on Western blotting of microsomal fractions from lipopolysaccharide-treated mouse peritoneal macrophages. Northern and Western blotting analyses revealed that mPGES-1 was induced together with cyclooxygenase-2 in mouse macrophages after treatment of the cells with lipopolysaccharide. Confocal immunofluorescence microscopy revealed that both mPGES-1 and cyclooxygenase-2 were colocalized in the lipopolysaccharide-treated macrophages. Taken together, these results demonstrate that mPGES-1 is an efficient downstream enzyme for the production of PGE2 in the activated macrophages treated by lipopolysaccharide.     

PGH2 degradation pathway catalyzed by GSH-heme complex bound microsomal prostaglandin E2 synthase type 2: the first example of a dual-function enzyme

Prostaglandin E2 synthase (PGES) catalyzes the isomerization of PGH2 to PGE2. PGES type 2 (mPGES-2) is a membrane-associated enzyme, whose N-terminal section is apparently inserted into the lipid bilayer. Both intact and N-terminal truncated enzymes have been isolated and have similar catalytic activity. The recombinant N-terminal truncated enzyme purified from Escherichia coli HB101 grown in LB medium containing delta-aminolevulinate and Fe(NO3)3 has a red color, while the same enzyme purified from the same E. coli grown in minimal medium has no color. The red-colored enzyme has been characterized by mass, fluorescence, and EPR spectroscopies and X-ray crystallography. The enzyme is found to contain bound glutathione (GSH) and heme. GSH binds to the active site with six H-bonds, while a heme is complexed with bound GSH forming a S-Fe coordination bond with no polar interaction with mPGES-2. There is a large open space between the heme and the protein, where a PGH2 might be able to bind. The heme dissociation constant is 0.53 microM, indicating that mPGES-2 has relatively strong heme affinity. Indeed, expression of mPGES-2 in E. coli stimulates heme biosynthesis. Although mPGES-2 has been reported to be a GSH-independent PGES, the crystal structure and sequence analysis indicate that mPGES-2 is a GSH-binding protein. The GSH-heme complex-bound enzyme (mPGES-2h) catalyzes formation of 12(S)-hydroxy-5(Z),8(E),10(E)-heptadecatrienoic acid and malondialdehyde from PGH2, but not formation of PGE2. The following kinetic parameters at 37 degrees C were determined: KM = 56 microM, kcat = 63 s-1, and kcat/KM = 1.1 x 10(6) M-1 s-1. They suggest that mPGES-2h has significant catalytic activity for PGH2 degradation. It is possible that both GSH-heme complex-free and -bound enzymes are present in the same tissues. mPGES-2 in heme-rich liver is most likely to become the form of mPGES-2h and might be involved in degradation reactions similar to that of cytochrome P450. Since mPGES-2 is an isomerase and mPGES-2h is a lyase, mPGES-2 cannot simply be classified into one of six classes set by the International Union of Biochemistry and Molecular Biology.     

HTRF-based assay for microsomal prostaglandin E2 synthase-1 activity

Microsomal prostaglandin E(2) synthase-1 (mPGES-1) catalyzes the formation of prostaglandin E(2) (PGE(2)) from the endoperoxide prostaglandin H( 2) (PGH(2)). Expression of this enzyme is induced during the inflammatory response, and mouse knockout experiments suggest it may be an attractive target for antiarthritic therapies. Assaying the activity of this enzyme in vitro is challenging because of the unstable nature of the PGH( 2) substrate. Here, the authors present an mPGES-1 activity assay suitable for characterization of enzyme preparations and for determining the potency of inhibitor compounds. This plate-based competition assay uses homogenous time-resolved fluorescence to measure PGE(2) produced by the enzyme. The assay is insensitive to DMSO concentration up to 10% and does not require extensive washes after the initial enzyme reaction is concluded, making it a simple and convenient way to assess mPGES-1 inhibition.     

Redirection of eicosanoid metabolism in mPGES-1-deficient macrophages

Microsomal prostaglandin E synthase (mPGES)-1 is one of several prostaglandin E synthases involved in prostaglandin H2 (PGH2) metabolism. In the present report, we characterize the contribution of mPGES-1 to cellular PGH2 metabolism in murine macrophages by studying the synthesis of eicosanoids and expression of eicosanoid metabolism enzymes in wild type and mPGES-1-deficient macrophages. Thioglycollate-elicited macrophages isolated from mPGES-1-/- animals and genetically matched wild type controls were stimulated with diverse pro-inflammatory stimuli. Prostaglandins were released in the following order of decreasing abundance from wild type macrophages stimulated with lipopolysaccharide: prostaglandin E2 (PGE2)>thromboxane B2 (TxB2)>6-keto prostaglandin F1alpha (PGF1alpha), prostaglandin F(2alpha) (PGF2alpha), and prostaglandin D2 (PGD2). In contrast, we detected in mPGES-1-/- macrophages a >95% reduction in PGE2 production resulting in the following altered prostaglandin profile: TxB2>6-keto PGF1alpha and PGF2alpha>PGE2, despite the comparable release of total prostaglandins. No significant change in expression pattern of key prostaglandin-synthesizing enzymes was detected between the genotypes. We then further profiled genotype-related differences in the eicosanoid profile using macrophages pre-stimulated with lipopolysaccharide followed by a 10-min incubation with 10 microm [3H]arachidonic acid. Eicosanoid products were subsequently identified by reverse phase high pressure liquid chromatography. The dramatic reduction in [3H]PGE2 formation from mPGES-1-/- macrophages compared with controls resulted in TxB2 and 6-keto PGF1alpha becoming the two most abundant prostaglandins in these samples. Our results also suggest a 5-fold increase in 12-[3H]hydroxyheptadecatrienoic acid release in mPGES-1-/- samples. Our data support the hypothesis that mPGES-1 induction in response to an inflammatory stimulus is essential for PGE2 synthesis. The redirection of prostaglandin production in mPGES-1-/- cells provides novel insights into how a cell processes the unstable endoperoxide PGH2 during the inactivation of a major metabolic outlet.     

Mutation of a critical arginine in microsomal prostaglandin E synthase-1 shifts the isomerase activity to a reductase activity that converts prostaglandin H2 into prostaglandin F2alpha

Microsomal prostaglandin E synthase type 1 (mPGES-1) converts prostaglandin endoperoxides, generated from arachidonic acid by cyclooxygenases, into prostaglandin E2. This enzyme belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral membrane proteins, and because of its link to inflammatory conditions and preferential coupling to cyclooxygenase 2, it has received considerable attention as a drug target. Based on the high resolution crystal structure of human leukotriene C4 synthase, a model of mPGES-1 has been constructed in which the tripeptide co-substrate glutathione is bound in a horseshoe-shaped conformation with its thiol group positioned in close proximity to Arg-126. Mutation of Arg-126 into an Ala or Gln strongly reduces the enzyme's prostaglandin E synthase activity (85-95%), whereas mutation of a neighboring Arg-122 does not have any significant effect. Interestingly, R126A and R126Q mPGES-1 exhibit a novel, glutathione-dependent, reductase activity, which allows conversion of prostaglandin H2 into prostaglandin F2alpha. Our data show that Arg-126 is a catalytic residue in mPGES-1 and suggest that MAPEG enzymes share significant structural components of their active sites.     

Purification and characterization of recombinant microsomal prostaglandin E synthase-1

Recombinant human microsomal prostaglandin E(2) synthase-1 (mPGES-1) was expressed in a baculovirus-Sf9 cell system. The mPGES-1 was solubilized from Sf9 cell membranes with diheptanoylphosphatidylcholine and purified in the presence of octylglucoside using hydroxyapatite column chromatography. The K(m) values of the substrates PGH(2) and GSH were 14 microM and 0.75 mM, respectively, with the purified enzyme. The specific activity (4 micromol/min/mg) was increased 3-5-fold by non-ionic and zwitterionic detergents. Kinetic analysis showed that dodecylmaltoside increases V(max) but does not affect the K(m) values of either substrate. Several other thiol-containing compounds were tested as glutathione replacements, none of which yielded detectable enzyme activity. During enzyme catalysis, glutathione was not oxidized and therefore can be considered an enzyme cofactor. No glutathione transferase or peroxidase activity could be determined with a range of potential substrates. The results show that purified mPGES-1 has a specific activity similar to Cox-2, consistent with its postulated role in Cox-2 mediated PGE(2) formation.     

Determination of prostaglandin E(2) by on-line solid-phase extraction-liquid chromatography with ultraviolet detection for microsomal prostaglandin E(2) synthase-1 inhibitor screening

A rapid, robust and selective on-line solid-phase extraction-liquid chromatographic method with ultra-violet detection (on-line SPE-LC-UV) for microsomal prostaglandin E(2) synthase-1 (mPGES-1) inhibitor screening was developed and validated. Disrupted A549 cells were used as mPGES-1 source and the formation of prostaglandin E(2) (PGE(2)) out of the substrate prostaglandin H(2) (PGH(2)) was determined at 195 nm. Direct on-line sample clean up was achieved by automated column switch (C18 trap column) prior isocratic separation using a C18 analytical column. The on-line SPE-LC-UV method was accurate, precise and reproducible in the range of 71-1763 ng/ml for PGE(2) and met the generally accepted criteria for bioanalytical methods. The method was successfully applied to determine the IC(50) value of the known mPGES-1 inhibitor NS-398.     

Novel human mPGES-1 inhibitors identified through structure-based virtual screening

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase after exposure to pro-inflammatory stimuli and, therefore, represents a novel target for therapeutic treatment of acute and chronic inflammatory disorders. It is essential to identify mPGES-1 inhibitors with novel scaffolds as new leads or hits for the purpose of drug design and discovery that aim to develop the next-generation anti-inflammatory drugs. Herein we report novel mPGES-1 inhibitors identified through a combination of large-scale structure-based virtual screening, flexible docking, molecular dynamics simulations, binding free energy calculations, and in vitro assays on the actual inhibitory activity of the computationally selected compounds. The computational studies are based on our recently developed three-dimensional (3D) structural model of mPGES-1 in its open state. The combined computational and experimental studies have led to identification of new mPGES-1 inhibitors with new scaffolds. In particular, (Z)-5-benzylidene-2-iminothiazolidin-4-one is a promising novel scaffold for the further rational design and discovery of new mPGES-1 inhibitors. To our best knowledge, this is the first time a 3D structural model of the open state mPGES-1 is used in structure-based virtual screening of a large library of available compounds for the mPGES-1 inhibitor identification. The positive experimental results suggest that our recently modeled trimeric structure of mPGES-1 in its open state is ready for the structure-based drug design and discovery.     

Observation of two modes of inhibition of human microsomal prostaglandin E synthase 1 by the cyclopentenone 15-deoxy-Δ(12,14)-prostaglandin J(2)

Microsomal prostaglandin E synthase 1 (MPGES1) is an enzyme that produces the pro-inflammatory molecule prostaglandin E(2) (PGE(2)). Effective inhibitors of MPGES1 are of considerable pharmacological interest for the selective control of pain, fever, and inflammation. The isoprostane, 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a naturally occurring degradation product of prostaglandin D(2), is known to have anti-inflammatory properties. In this paper, we demonstrate that 15d-PGJ(2) can inhibit MPGES1 by covalent modification of residue C59 and by noncovalent inhibition through binding at the substrate (PGH(2)) binding site. The mechanism of inhibition is dissected by analysis of the native enzyme and the MPGES1 C59A mutant in the presence of glutathione (GSH) and glutathione sulfonate. The location of inhibitor adduction and noncovalent binding was determined by triple mass spectrometry sequencing and with backbone amide H/D exchange mass spectrometry. The kinetics, regiochemistry, and stereochemistry of the spontaneous reaction of GSH with 15d-PGJ(2) were determined. The question of whether the anti-inflammatory properties of 15d-PGJ(2) are due to inhibition of MPGES1 is discussed.     

Lead discovery for microsomal prostaglandin E synthase using a combination of high-throughput fluorescent-based assays and RapidFire mass spectrometry

Microsomal prostaglandin E synthase-1 (mPGES-1) represents an attractive target for the treatment of rheumatoid arthritis and pain, being upregulated in response to inflammatory stimuli. Biochemical assays for prostaglandin E synthase activity are complicated by the instability of the substrate (PGH(2)) and the challenge of detection of the product (PGE(2)). A coupled fluorescent assay is described for mPGES-1 where PGH(2) is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE(2) is detected by coupling through 15-prostaglandin dehydrogenase (15-PGDH) and diaphorase. The overall coupled reaction was miniaturized to 1536-well plates and validated for high-throughput screening. For compound progression, a novel high-throughput mass spectrometry assay was developed using the RapidFire platform. The assay employs the same in situ substrate generation step as the fluorescent assay, after which both PGE(2) and a reduced form of the unreacted substrate were detected by mass spectrometry. Pharmacology and assay quality were comparable between both assays, but the mass spectrometry assay was shown to be less susceptible to interference and false positives. Exploiting the throughput of the fluorescent assay and the label-free, direct detection of the RapidFire has proved to be a powerful lead discovery strategy for this challenging target.     

Characterization of the substrate mimic bound to engineered prostacyclin synthase in solution using high-resolution NMR spectroscopy and mutagenesis: implication of the molecular mechanism in biosynthesis of prostacyclin

High-resolution NMR spectroscopy was used to determine the docking of a substrate (prostaglandin H2) mimic (U46619) to the engineered prostacyclin (PGI2) synthase (PGIS) in solution. The binding of U46619 to the PGIS protein was demonstrated by 1D NMR titration, and the significant perturbation of the chemical shifts of protons at C-11, H2C, and H20 of U46619 were observed upon U46619 binding to the engineered PGIS in a concentration-dependent manner. The detailed conformational change and 3D structure of the PGIS-bound U46619 were further demonstrated by 2D 1H NMR experiments using the transferred NOE technique. The distances between the protons H20 and H2, H18 and H2, and H18 and H4 are shorter following their binding to the PGIS in solution-down to within 5 A. These shorter distances resulted in a widely open conformation, where the triangle shape of the unbound U46619 changed to a more compact conformation with an oval shape. The bound conformation of U46619 fits the crystal structure of the PGIS substrate binding pocket considerably better than that of the unbound U46619. The residues important to the substrate binding in the active site pocket of PGIS were also predicted. For example, Trp282 could be one of the most important residues and is suspected to play a role in the determination of specific catalytic function, which has been established by the docking studies using the NMR structure of the PGIS-bound form of U46619 and the PGIS crystal structure. These studies have provided the structural information for the interaction of the PGIS with its substrate mimic. The noted conformational changes where the C-6 position is closer to the C-9 position of U46619 provided the first experimental data for understanding the molecular mechanism of the catalytic function of PGIS in the isomerization of PGH2 to prostacyclin.     

Intracellular-specific colocalization of prostaglandin E2 synthases and cyclooxygenases in the brain

Prostaglandin E2 (PGE2) is the major primary prostaglandin generated by brain cells. However, the coordination and intracellular localization of the cyclooxygenases (COXs) and prostaglandin E synthases (PGESs) that convert arachidonic acid to PGE2 in brain tissue are not known. We aimed to determine whether microsomal and cytosolic PGES (mPGES-1 and cPGES) colocalize and coordinate activity with either COX-1 or COX-2 in brain tissue, particularly during development. Importantly, we found that cytosolic PGES also associates with microsomes (cPGES-m) from the cerebrum and cerebral vasculature of the pig and rat as well as microsomes from various cell lines; this seemed dependent on the carboxyl terminal 35-amino acid domain and a cysteine residue (C58) of cPGES. In microsomal membranes from the postnatal brain and cerebral microvessels of mature animals, cPGES-m colocalized with both COX-1 and COX-2, whereas mPGES-1 was undetectable in these microsomes. Accordingly, in this cell compartment, cPGES could coordinate its activity with COX-2 and COX-1 (partly inhibited by NS398); albeit in microsomes of the brain microvasculature from newborns, mPGES-1 was also present. In contrast, in nuclei of brain parenchymal and endothelial cells, mPGES-1 and cPGES colocalized exclusively with COX-2 (determined by immunoblotting and immunohistochemistry); these PGESs contributed to conversion of PGH2 into PGE2. Hence, contrary to a previously proposed model of exclusive COX-2/mPGES-1 coordination, COX-2 can coordinate with mPGES-1 and/or cPGES in the brain, depending on the cell compartment and the age group.     

Solution structure of a common substrate mimetic of cyclooxygenase-downstream synthases bound to an engineered thromboxane A2 synthase using a high-resolution NMR technique

Understanding the docking mechanism of the common substrate, prostaglandin H(2) (PGH(2)), into the active sites of different cyclooxygenase(COX)-downstream synthases is a key step toward uncovering the molecular basis of the isomerization of PGH(2) to different prostanoids. A high-resolution NMR spectroscopy was used to determine the conformational changes and solution 3D structure of U44069, a PGH(2) analogue, bound to one of the COX-downstream synthases-an engineered thromboxane A(2) synthase (TXAS). The dynamic binding was clearly observed by (1)D NMR titration. The detailed conformational change and 3D structure of U44069 bound to the TXAS were demonstrated by 2D (1)H NMR experiments using transferred NOEs. Through the assignments for the 2D (1)H NMR spectra, TOCSY, DQF-COSY, NOESY, and the structural calculations based on the NOE constraints, they demonstrated that the widely open conformation with a triangle shape of the free U44069 changed to a compact structure with an oval shape when bound to the TXAS. The putative substrate-binding pocket of the TXAS model fits the conformation of the TXAS-bound U44069 appropriately, but could not fit the free form of U44069. It was the first to provide structural information for the dynamic docking of the PGH(2) mimic of the TXAS in solution, and to imply that PGH(2) undergoes conformational changes when bound to different COX-downstream synthases, which may play important roles in the isomerization of PGH(2) to different prostanoids. The NMR technique can be used as a powerful tool to determine the conformations of PGH(2) bound to other COX-downstream synthases.     

Liver X receptor ligands inhibit the lipopolysaccharide-induced expression of microsomal prostaglandin E synthase-1 and diminish prostaglandin E2 production in murine peritoneal macrophages

Microsomal prostaglandin E synthase (mPGES)-1, which is dramatically induced in macrophages by inflammatory stimuli such as lipopolysaccharide (LPS), catalyzes the conversion of cyclooxygenase-2 (COX-2) reaction product prostaglandin H(2) (PGH(2)) into prostaglandin E(2) (PGE(2)). The mPGES-1-derived PGE(2) is thought to help regulate inflammatory responses. On the other hand, excess PGE(2) derived from mPGES-1 contributes to the development of inflammatory diseases such as arthritis and inflammatory pain. Here, we examined the effects of liver X receptor (LXR) ligands on LPS-induced mPGES-1 expression in murine peritoneal macrophages. The LXR ligands 22(R)-hydroxycholesterol (22R-HC) and T0901317 reduced LPS-induced expression of mPGES-1 mRNA and mPGES-1 protein as well as that of COX-2 protein. However, LXR ligands did not influence the expression of microsomal PGES-2 (mPGES-2) or cytosolic PGES (cPGES) protein. Consequently, LXR ligands suppressed the production of PGE(2) in macrophages. These results suggest that LXR ligands diminish PGE(2) production by inhibiting the LPS-induced gene expression of the COX-2-mPGES-1 axis in LPS-activated macrophages.     

Structural and functional characterization of HQL-79, an orally selective inhibitor of human hematopoietic prostaglandin D synthase

We determined the crystal structure of human hematopoietic prostaglandin (PG) D synthase (H-PGDS) as the quaternary complex with glutathione (GSH), Mg2+, and an inhibitor, HQL-79, having anti-inflammatory activities in vivo, at a 1.45-A resolution. In the quaternary complex, HQL-79 was found to reside within the catalytic cleft between Trp104 and GSH. HQL-79 was stabilized by interaction of a phenyl ring of its diphenyl group with Trp104 and by its piperidine group with GSH and Arg14 through water molecules, which form a network with hydrogen bonding and salt bridges linked to Mg2+. HQL-79 inhibited human H-PGDS competitively against the substrate PGH2 and non-competitively against GSH with Ki of 5 and 3 microm, respectively. Surface plasmon resonance analysis revealed that HQL-79 bound to H-PGDS with an affinity that was 12-fold higher in the presence of GSH and Mg2+ (Kd, 0.8 microm) than in their absence. Mutational studies revealed that Arg14 was important for the Mg2+-mediated increase in the binding affinity of H-PGDS for HQL-79, and that Trp104, Lys112, and Lys198 were important for maintaining the HQL-binding pocket. HQL-79 selectively inhibited PGD2 production by H-PGDS-expressing human megakaryocytes and rat mastocytoma cells with an IC50 value of about 100 microm but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between H-PGDS and cyclooxygenase. Orally administered HQL-79 (30 mg/kg body weight) inhibited antigen-induced production of PGD2, without affecting the production of PGE2 and PGF2alpha, and ameliorated airway inflammation in wild-type and human H-PGDS-overexpressing mice. Knowledge about this structure of quaternary complex is useful for understanding the inhibitory mechanism of HQL-79 and should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit PGD2 production specifically.     

Cyclin-dependent kinase 5 phosphorylates mammalian HMGB1 protein only if acetylated

Old yellow enzyme (OYE) is an NADPH oxidoreductase capable of reducing a variety of compounds. It contains flavin mononucleotide (FMN) as a prosthetic group. A ternary complex structure of OYE from Trypanosoma cruzi (TcOYE) with FMN and one of the substrates, p-hydroxybenzaldehyde, shows a striking movement around the active site upon binding of the substrate. From a structural comparison of other OYE complexed with 12-oxophytodienoate, we have constructed a complex structure with another substrate, prostaglandin H(2) (PGH(2)), to provide a proposed stereoselective reaction mechanism for the reduction of PGH(2) to prostaglandin F(2α) by TcOYE.     

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.