UV resonance Raman studies on the activation mechanism of human hematopoietic prostaglandin D2 synthase by a divalent cation, Mg2+
Introduction
Hematopoietic prostaglandin (PG) D2 synthase (H-PGDS) catalyzes the isomerization of prostaglandin H2 (PGH2) to PGD2 in the presence of glutathione (GSH) in a highly specific manner [1]. PGD2 acts as an allergic and inflammatory mediator and participates in the inhibition of platelet aggregation and the induction of vasodilation and bronchoconstriction [2], [3], [4]. Overproduction of PGD2 has been reported to exacerbate asthmatic response [5]. H-PGDS was first purified from a rat spleen and identified to be the only vertebrate σ-class glutathione S-transferase (GST) [6], [7]. Very recently, the activation of human H-PGDS by such divalent metal ions as Ca2+ and Mg2+ has been reported by Urade and coworkers [8].
X-ray crystallography revealed that the metal ion binds to the dimer interface of H-PGDS through six asparagic acid residues (Asp93, Asp96, and Asp97 from each subunit) (Fig. 1A). H-PGDS is a homodimeric enzyme with 23 kDa subunits. Each H-PGDS subunit is comprised of the N-terminal domain containing a four-stranded β sheet and three α helices, and of the C-terminal domain containing five α helices (Fig. 1B). There are two dominant active sites, a GSH binding site (G-site) and a hydrophobic substrate binding site (H-site) between the two domains [8], [9]. Site-directed mutagenesis of rat H-PGDS has demonstrated that Tyr8, Arg14 and Trp104 were essential for the catalytic reaction [10]. Especially, Tyr8 and Arg14 are considered to be very important for expressing the enzymatic activity of GSH. However, the detailed function of these residues remains unclear.
GSTs are multifunctional enzymes playing important roles in the cellular detoxification processes. These enzymes catalyze the conjugation of GSH to a wide variety of electrophilic compounds such as xenobiotic and endogenous toxicants [11]. A catalytic tyrosine residue at the G-site is conserved among the α-, μ-, and π-class GSTs. The 3-D structures of these GSTs with GSH, substrate analogue, or the product demonstrate that the hydroxyl groups of the conserved tyrosine residues lie within a hydrogen-bonding distance of the sulfur atom of GSH [12]. Electronic absorption spectroscopic studies suggest that the GSTs effectively lower the pKa (6.2–6.8) of the GSH thiol group, resulting in the formation of the nucleophile thiolate at physiological pH [13], [14], [15], [16]. Atkins et al. have reported that the hydroxyl group of the essential G-site Tyr9 has an unusually low pKa value (8.3–8.5) in the α-class GST, and suggested that the tyrosinate acts as a general base (TyrO−-HSG) to abstract H+ from the GSH thiol group [17]. On the other hand, Armstrong et al. have suggested that the catalytic G-site Tyr6 in the μ-class GST has a normal pKa, and that the hydroxyl group of Tyr6 stabilizes the thiolate anion of GSH through a hydrogen bond (TyrOH--GS) during the catalytic reaction of the μ-class GST [15], [18].
Ultraviolet resonance Raman (UVRR) spectroscopy is a powerful technique to investigate aromatic amino acid residues in proteins. The recent developments of stable lasers and sensitive detectors have made it possible to use the UVRR spectroscopy for the analysis of protein structures and functions [19], [20], [21]. A strong intensity enhancement is expected for the vibrational modes associated with tryptophan and tyrosine residues by the excitation in the deep-UV region [22], [23], [24], [25]. The UVRR spectrum of tyrosine excited at 244 nm contains several vibrational marker bands that provide useful probes of the protein structural aspects. The frequencies and intensities of these marker bands have been successfully used to examine the protein structure, hydrogen bonding, and environmental effects [26], [27], [28]. p-Cresol has been used as a model compound of the tyrosine phenol group, because the vibrational patterns of tyrosine and p-cresol are similar [26], [27], [29].
Asher et al. have applied the correlation between the Y8a Raman band intensity and pH to determine the pKa value of the tyrosine phenol group in horse and sperm whale myoglobins [30]. UVRR spectra of sperm whale myoglobin demonstrated that the structural change of Tyr151 occurs upon binding of CO to the heme active site [31]. UVRR spectra of apo-human serum transferrin demonstrated that a low pKa of tyrosine residue was elevated upon binding of sulfate [32]. In the allosteric pathway of hemoglobin, UVRR spectroscopy has revealed that the hydrogen bonding of tyrosine with several other residues is involved in the dynamics of the R-T transition [33], [34], [35], [36].
In this study, UVRR spectroscopic analyses of H-PGDS in the absence and presence of Mg2+ and GSH were performed in order to elucidate the long range activation mechanisms of H-PGDS by a divalent cation at the enzyme dimmer interface. This is the first report on the UVRR analyses of the GST superfamily involving hematopoietic prostaglandin D2 synthase.
Section snippets
Materials
Reagents used in this study were obtained from the following sources: glutathione (GSH) and 1-chrolo-2,4-dinitrobenzene (CDNB) from Sigma; GSH Sepharose 4B column and superdex 75 pg column from GE Healthcare Bioscience.
Expression and purification of the recombinant protein
The plasmid pT7-7 vector inserted human H-PGDS cDNA or Tyr8Phe mutant cDNA (Osaka Bioscience Institute) was introduced into E. coli BL21 (DE3). The transformant was cultivated in LB-ampicillin (0.1 mg/mL) medium. When OD600 of the cultivate medium reached 0.6–0.8, 0.4 mM,
Effect of Mg2+ on the conjugation reaction of GSH to CDNB by H-PGDS and its Tyr8Phe mutant
The conjugation of GSH and CDNB catalyzed by H-PGDS in the absence or presence of Mg2+ were investigated for the wild-type enzyme and its Tyr8Phe mutant. Fig. 2 shows the plots for the initial conjugation reaction rates of H-PGDS and Tyr8Phe versus GSH concentration. The respective kinetic parameters, Michaelis constants for GSH (), maximum velocities (Vmax), and catalytic rate constants (kcat) were obtained (see Eqs. (1), (2) in Experimental Section) as summarized in Table 1. The Vmax
Discussion
The tyrosine residues conserved at the G-site of several GSTs (Tyr8 of αGST1-1, Tyr9 of GST YaYa, Tyr7 of πGST, and Tyr6 of μGST3-3) have been reported to be important for the enzymatic reaction [15], [16], [49], [50]. These conserved tyrosine residues are considered to activate or stabilize a GST cofactor, GSH [15], [17], [18]. Human H-PGDS, which is identified to be an isozyme of the σ-class GST, also has the conserved tyrosine residue at the G-site, Tyr8 [9]. This Tyr8 residue has been
Acknowledgments
We are grateful to Prof. Roman S. Czernuszewicz for careful reading of the manuscript and valuable suggestions. This work was supported in part by the following: Project of Development of Basic Technologies for Advanced Production Methods Using Microorganism Functions by the New Energy and Industrial Technology Development Organization (NEDO) to TK and a Grants-in-Aid No. 19550004 (to SM) for Scientific Research from JSPS. The generous allotment of computational time from the Research Center
References (56)
- et al.
J. Biol. Chem.
(1987) - et al.
Prostalgandins
(1978) - et al.
Biochim. Biophys. Acta
(1979) - et al.
Cell
(1997) - et al.
J. Biol. Chem.
(2000) - et al.
J. Biol. Chem.
(1992) - et al.
J. Biol. Chem.
(1992) - et al.
J. Biol. Chem.
(1993) Prog. Biophys. Mol. Biol.
(1992)- et al.
Methods Enzymol.
(1993)
Spectrochimica Acta
FEBS Letters
J. Mol. Biol.
J. Mol. Biol.
J. Biol. Chem.
FEBS Lett.
Biochem. Biophys. Res. Commun.
Structure
J. Mol. Biol.
Science
Eur. Respir. J.
J. Immunol.
Eur. J. Biol.
Natl. Struct. Biol.
Crit. Rev. Biochem. Mol. Biol.
Eur. J. Biochem.
Biochemistry
Biochemistry
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