FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2082079362> ?p ?o ?g. }

• W2082079362 endingPage "18244" @default.
• W2082079362 startingPage "18233" @default.
• W2082079362 abstract "The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic “dome” over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site. The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic “dome” over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site. Prostaglandin endoperoxide H synthases (PGHSs) 2The abbreviations used are: PGHS, prostaglandin (PG) endoperoxide H synthase; huPGHS, human PGHS; ovPGHS, ovine PGHS; muPGHS, murine PGHS; COX, cyclooxygenase; POX, peroxidase; PPIX, protoporphyrin IX; PG, prostaglandin; HPETE, hydroperoxyeicosatetraenoic acid; EtOOH, ethyl hydroperoxide; H2O2, hydrogen peroxide; t-BuOOH, t-butyl hydroperoxide; HETE, hydroxyeicosatetraenoic; KETE, ketoeicosatetraenoic acid; PPA, 5-phenyl-4-pentenyl alcohol; HPLC, high performance liquid chromatography; BDE, bond dissociation energy; MD, Molecular Dynamics; Ni-NTA, nickel-nitrilotriacetic acid; PPIX, protoporphyrin IX. 2The abbreviations used are: PGHS, prostaglandin (PG) endoperoxide H synthase; huPGHS, human PGHS; ovPGHS, ovine PGHS; muPGHS, murine PGHS; COX, cyclooxygenase; POX, peroxidase; PPIX, protoporphyrin IX; PG, prostaglandin; HPETE, hydroperoxyeicosatetraenoic acid; EtOOH, ethyl hydroperoxide; H2O2, hydrogen peroxide; t-BuOOH, t-butyl hydroperoxide; HETE, hydroxyeicosatetraenoic; KETE, ketoeicosatetraenoic acid; PPA, 5-phenyl-4-pentenyl alcohol; HPLC, high performance liquid chromatography; BDE, bond dissociation energy; MD, Molecular Dynamics; Ni-NTA, nickel-nitrilotriacetic acid; PPIX, protoporphyrin IX. catalyze the committed step in prostaglandin (PG) formation (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2394) Google Scholar, 2Kulmacz R.J. van der Donk W.A. Tsai A.L. Prog. Lipid Res. 2003; 42: 377-404Crossref PubMed Scopus (75) Google Scholar, 3Rouzer C. Marnett L. Chem. Rev. 2003; 103: 2239-2304Crossref PubMed Scopus (195) Google Scholar). There are two PGHS isoforms, PGHS-1 and PGHS-2, which are also known as cyclooxygenase-1 and -2 (COX-1 and -2). PGHSs catalyze two reactions including a COX reaction in which arachidonic acid is converted to prostaglandin G2 (PGG2) and a peroxidase (POX) reaction in which PGG2 is reduced to PGH2 (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2394) Google Scholar, 2Kulmacz R.J. van der Donk W.A. Tsai A.L. Prog. Lipid Res. 2003; 42: 377-404Crossref PubMed Scopus (75) Google Scholar, 3Rouzer C. Marnett L. Chem. Rev. 2003; 103: 2239-2304Crossref PubMed Scopus (195) Google Scholar). Both PGHS-1 and PGHS-2 are located on the luminal side of the endoplasmic reticulum and nuclear envelope (4Rollins T.E. Smith W.L. J. Biol. Chem. 1980; 255: 4872-4875Abstract Full Text PDF PubMed Google Scholar, 5Smith W.L. Rollins T.E. DeWitt D.L. Prog. Lipid Res. 1981; 20: 103-110Crossref PubMed Scopus (11) Google Scholar, 6Spencer A.G. Woods J.W. Arakawa T. Singer I.I. Smith W.L. J. Biol. Chem. 1998; 273: 9886-9893Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 7Otto J.C. Smith W.L. Adv. Prostaglandin Thromboxane Leukotriene Res. 1995; 23: 29-34PubMed Google Scholar). In general, PGHS-1 is constitutively expressed, whereas PGHS-2 is inducibly expressed in many cell types (8Tanabe T. Tohnai N. Prostaglandins Other Lipid Mediat. 2002; 68–69: 95-114Crossref PubMed Scopus (365) Google Scholar, 9Kang Y.-J. Mbonye U.R. DeLong C.J. Wada M. Smith W.L. Prog. Lipid Res. 2007; 46: 108-125Crossref PubMed Scopus (224) Google Scholar). The COX and POX reactions occur at structurally distinct but functionally interconnected sites of PGHSs (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2394) Google Scholar, 2Kulmacz R.J. van der Donk W.A. Tsai A.L. Prog. Lipid Res. 2003; 42: 377-404Crossref PubMed Scopus (75) Google Scholar, 3Rouzer C. Marnett L. Chem. Rev. 2003; 103: 2239-2304Crossref PubMed Scopus (195) Google Scholar). In the POX reaction the Fe3+ protoporphyrin IX (PPIX) is first oxidized to an oxyferryl heme radical π -cation, referred to as Compound I, which is similar to Compound I of horseradish peroxidase (10Schulz C.E. Rutter R. Sage J.T. Debrunner P.G. Hager L.P. Biochemistry. 1984; 23: 4743-4754Crossref PubMed Scopus (217) Google Scholar, 11Ortiz de Montellano P. Cytochrome P450: Structure, Mechanism and Biochemistry. Plenum Press, New York1995: 49-80Crossref Google Scholar, 12Patterson W.R. Poulos T.L. Goodin D.B. Biochemistry. 1995; 34: 4342-4345Crossref PubMed Scopus (162) Google Scholar, 13Benecky M.J. Frew J.E. Scowen N. Jones P. Hoffman B.M. Biochemistry. 1993; 32: 11929-11933Crossref PubMed Scopus (91) Google Scholar). Compound I can either be reduced by exogenous reductants to an oxyferryl heme form (Compound II), or it can undergo intramolecular reduction, involving transfer of an electron from Tyr-385, forming a Compound II-like spectral intermediate and a tyrosyl radical (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2394) Google Scholar, 2Kulmacz R.J. van der Donk W.A. Tsai A.L. Prog. Lipid Res. 2003; 42: 377-404Crossref PubMed Scopus (75) Google Scholar, 3Rouzer C. Marnett L. Chem. Rev. 2003; 103: 2239-2304Crossref PubMed Scopus (195) Google Scholar, 14Karthein R. Dietz R. Nastainczyk W. Ruf H.H. Eur. J. Biochem. 1988; 171: 313-320Crossref PubMed Scopus (230) Google Scholar, 15Dietz R. Nastainczyk W. Ruf H.H. Eur. J. Biochem. 1988; 171: 321-328Crossref PubMed Scopus (194) Google Scholar). This latter complex is known as Intermediate II and is analogous to the intermediate ES of cytochrome c peroxidase (16Erman J.E. Vitello L.B. Mauro J.M. Kraut J. Biochemistry. 1989; 28: 7992-7995Crossref PubMed Scopus (178) Google Scholar, 17Houseman A.L. Doan P.E. Goodin D.B. Hoffman B.M. Biochemistry. 1993; 32: 4430-4443Crossref PubMed Scopus (104) Google Scholar, 18Huyett J.E. Choudhury S.B. Eichhorn D.M. Bryngelson P.A. Maroney M.J. Hoffman B.M. Inorg. Chem. 1998; 37: 1361-1367Crossref PubMed Scopus (61) Google Scholar, 19Sivaraja M. Goodin D.B. Smith M. Hoffman B.M. Science. 1989; 245: 738-740Crossref PubMed Scopus (469) Google Scholar). Intermediate II is the COX active form of the enzyme. When arachidonic acid is present in the COX site, the Tyr-385 radical removes a hydrogen atom from C-13 of arachidonate, triggering the COX reaction (20Hamberg M. Samuelsson B. J. Biol. Chem. 1967; 242: 5336-5343Abstract Full Text PDF PubMed Google Scholar). Intermediate II is regenerated within the COX site when PGG2 is produced. PGHS reconstituted with Mn3+-PPIX proceeds through a POX catalytic cycle analogous to that of Fe3+-PPIX PGHS, but formation of the mangano-Compound I-like species is much slower (21Tsai A. Wei C. Baek H.K. Kulmacz R.J. Van Wart H.E. J. Biol. Chem. 1997; 272: 8885-8894Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 22Landino L.M. Marnett L.J. Biochemistry. 1996; 35: 2637-2643Crossref PubMed Scopus (16) Google Scholar, 23Strieder S. Schaible K. Scherer H.J. Dietz R. Ruf H.H. J. Biol. Chem. 1992; 267: 13870-13878Abstract Full Text PDF PubMed Google Scholar). Activation of the COX activity of PGHS-2 is triggered at a hydroperoxide concentration that is about 10 times lower than that required for PGHS-1 (24Lu G. Tsai A.L. Van Wart H.E. Kulmacz R.J. J. Biol. Chem. 1999; 274: 16162-16167Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). This may be important in cells co-expressing PGHS-1 and PGHS-2, where PGHS-2 COX activity can function when PGHS-1 COX activity is latent (25Reddy S.T. Herschman H.R. J. Biol. Chem. 1994; 269: 15473-15480Abstract Full Text PDF PubMed Google Scholar, 26Shitashige M. Morita I. Murota S. Biochim. Biophys. Acta. 1998; 1389: 57-66Crossref PubMed Scopus (70) Google Scholar). PGG2 is considered to be a natural substrate for PGHS POX activity, but other peroxides are also substrates. The identity of the peroxide that initiates COX activity in vivo is not known. PGHS-1 is reported to prefer primary and secondary alkyl hydroperoxides to H2O2 or bulky peroxides like t-butyl hydroperoxide (t-BuOOH). The molecular basis for this substrate preference is not known. Examination of the crystal structures of the POX active sites in ovine (ov) PGHS-1 (Fig. 1) and murine PGHS-2 indicate that the distal surface of the heme group is open to the solvent and that the site is sufficiently spacious to accommodate large and linear hydroperoxides coming directly from the aqueous environment (27Malkowski M.G. Ginell S.L. Smith W.L. Garavito R.M. Science. 2000; 289: 1933-1937Crossref PubMed Scopus (256) Google Scholar, 28Picot D. Loll P.J. Garavito R.M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1135) Google Scholar). A dome comprised of mostly hydrophobic amino acids lies above the distal surface of the heme, and Molecular Dynamics modeling has suggested that these residues can interact with alkyl chains of alkyl hydroperoxides related to PGG2 (29Seibold S.A. Smith W.L. Cukier R.I. J. Phys. Chem. B. 2004; 108: 9297-9305Crossref Scopus (5) Google Scholar, 30Chubb A.J. Fitzgerald D.J. Nolan K.B. Moman E. Biochemistry. 2006; 45: 811-820Crossref PubMed Scopus (15) Google Scholar). His-388 covalently binds to a high spin state iron in the heme plane as the fifth ligand, and a water molecule is thought to be the sixth heme ligand (31Seibold S.A. Cerda J.F. Mulichak A.M. Song I. Garavito R.M. Arakawa T. Smith W.L. Babcock G.T. Biochemistry. 2000; 39: 6616-6624Crossref PubMed Scopus (27) Google Scholar). Here we report studies designed to examine the molecular basis for the hydroperoxide substrate specificity of oPGHS-1. We first developed an improved method for obtaining steady state catalytic constants for PGHS POX activity that we used to quantify hydroperoxide substrate specificity. We then investigated a number of PGHS POX mutants to determine whether specific amino acids in the dome of the POX site are responsible for PGHS POX specificity. As part of these studies we reinvestigated the role of the distal glutamine (Gln-203) in catalysis. Finally, because we have found in previous studies that there is cross-talk between the COX sites of the monomers comprising PGHS dimers (32Yuan C. Rieke C.J. Rimon G. Wingerd B.A. Smith W.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6142-6147Crossref PubMed Scopus (98) Google Scholar), we performed experiments to determine whether there is cross-talk between the POX and COX sites of partner monomers. Materials—15-Hydroperoxyeicosatetraenoic acid (15-HPETE) was synthesized as described previously (33Graff G. Methods Enzymol. 1982; 86: 386-392Crossref Scopus (39) Google Scholar) or purchased from Cayman Chemical Company. Ethyl hydroperoxide (EtOOH) was from Polyscience. t-BuOOH, H2O2, guaiacol, phenol, and NaCN were from Sigma. Polyoxyethylene-6-decyl ether (C10E6) was from Anatrace. Fe3+-protoporphyrin 1X (PPIX) and Mn3+-PPIX were from Frontier Scientific. Arachidonic acid, 15-hydroxyeicosatetraenoic acid (15-HETE), 15-ketoeicosatetraenoic acid (15-KETE), and 5-phenyl-4-pentenyl alcohol (PPA) were from Cayman Chemical Co.. Hexanes, isopropanol, and acetic acid were HPLC grade from Fisher. Other chemicals were analytical grade from Sigma. Mutagenesis, Protein Expression, and Purification—A cDNA for ovPGHS-1 containing a hexahistidine (His6) tag at the N terminus (34Smith T. Leipprandt J. DeWitt D. Arch. Biochem. Biophys. 2000; 375: 195-200Crossref PubMed Scopus (54) Google Scholar, 35Song I. Ball T.M. Smith W.L. Biochem. Biophys. Res. Commun. 2001; 289: 869-875Crossref PubMed Scopus (37) Google Scholar) was subcloned into pFastBac plasmid (Invitrogen). The QuikChange site-directed mutagenesis protocol (Stratagene) was used to construct the mutants. pFastBac plasmids were used for transposition of DH10Bac Escherichia coli cells following Bac-to-Bac expression system protocols (Invitrogen). Mutants were identified by antibiotic resistance and blue/white screening. DNA was isolated and used to transfect Spodoptera frugiperda (Sf-21) insect cells (Invitrogen) as a Cellfectin (Invitrogen) lipid-bacmid DNA complex. Baculovirus was precipitated from media with polyethylene glycol (Mr 3350), and the DNA fragments containing mutant PGHSs were amplified by PCR for further sequence verification. Media containing baculovirus with correctly mutated sites were harvested and used for cell infection. Sf-21 cells were infected with a multiplicity of infection of 0.01, and cell pellets were harvested 4 days later when cell viability had dropped below 85%. Cell pellets were resuspended in 20 mm Tris-HCl, pH 8.0, and broken by sonication. Cell lysates were solubilized for 1 h with 0.8% C10E6. The supernatant after ultracentrifugation at 158,000 × g x 2 h was incubated with 4 ml of Ni-NTA fast flow-agarose (Qiagen) per liter of cell culture in the presence of 5% glycerol, 500 mm NaCl, and 5 mm imidazole. The slurry was poured into a column and washed with washing buffer (20 mm Tris-HCl, pH 8.0, 5% glycerol, 500 mm NaCl, 20 mm imidazole, and 0.1% C10E6). Bound PGHS was eluted with three column volumes of 250 mm imidazole. These latter eluates were pooled, concentrated, and desalted using a Sephadex G-25 (Sigma) spin column, which was pre-swelled in desalting buffer (20 mm Tris-HCl, pH 8.0, 5% glycerol, 150 NaCl and 0.02% C10E6). A G533A/Q203R-huPGHS-2 heterodimer was constructed and expressed using procedures described previously (32Yuan C. Rieke C.J. Rimon G. Wingerd B.A. Smith W.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6142-6147Crossref PubMed Scopus (98) Google Scholar). Mutated FLAG-tagged G533A-huPGHS-2 cDNA was cleaved from the pFastBac vector with StuI and KpnI and inserted downstream of Promoter p10 in pFastBac Dual vector that had been treated with SmaI and KpnI. Mutated His6-tagged Q203R-huPGHS-2 was cloned into Promoter PH of pFastBac Dual using the same strategy except that the EcoRI and HindIII were used to digest the DNA. The correct orientation and positions of the inserts were confirmed by sequencing and restriction digestion. The heterodimer was expressed in the baculovirus system as described above and purified by a combination of Ni-NTA and anti-FLAG-agarose chromatography (32Yuan C. Rieke C.J. Rimon G. Wingerd B.A. Smith W.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6142-6147Crossref PubMed Scopus (98) Google Scholar). The purity was determined by SDS-PAGE and by Western blot analysis using anti-His6 and anti-FLAG antibodies (Sigma). POX Activity Assays—POX reactions were conducted in 100 μl of filtered and degassed buffer, 100 mm Tris-HCl, pH 8.0, and 100 mm NaCl. Heme-reconstituted PGHS (76 nm) containing 9.0 mm guaiacol was mixed with an equal volume of a peroxide substrate solution using a stopped-flow apparatus (SX-60 HiTech Instruments). Formation of the guaiacol oxidation product 3,3′-dimethoxydipheno-4,4′-quinone was monitored at 436 nm (ϵ436 = 6,390 m–1 cm–1 (36Putter J. Methods of Enzymatic Analysis. Verlag Chemie, Weinheim, Germany1975: 685-690Google Scholar). The initial velocity v was determined when guaiacol oxidation was linear with time. Plots of the initial velocities as a function of hydroperoxide substrate concentrations were used in the Michaelis-Menten equation v = Vmax[S]/(Km + [S]) to determine Vmax and Km values; kcat is the activity/mol of enzyme. COX Activity Assays—COX reaction mixtures contained 3 ml of 0.1 m Tris-HCl, pH 8.0, 100 μm arachidonic acid, 1 mm phenol, and 5 μm hematin equilibrated in a glass chamber at 37 °C. Reactions were initiated by adding enzyme to the assay chamber. A Yellow Springs Instruments Model 53 oxygen monitor was used to monitor O2 consumption by native or mutant PGHSs with kinetic traces recorded using DasyLab (DasyTec) software. The rates reported are maximal rates occurring after a lag phase. One unit of COX activity is defined as 1 μmol of O2 consumed/min/mg of enzyme at 37 °C in the assay mixture. The lag time is defined as the time required for the COX activity to reach a maximum after initiating the reaction. Identification of POX Spectral Intermediates and Kinetic Analysis—Presteady state analysis of the POX reactions was performed with a rapid mixing and scanning technique using a stopped-flow apparatus equipped with double grating monochromators (DX-60 HiTech Instruments). ApoPGHS was reconstituted with Fe3+-PPIX or Mn3+-PPIX at a stoichiometry of 0.8 per enzyme monomer. The final heme concentrations were usually 1–2 μm, and substrate concentrations were 2–4 μm 15-HPETE or 50–100 μm H2O2. In single wavelength experiments at least 10-fold higher substrate concentrations were used to ensure steady state kinetics. Both the enzyme and substrate solutions were prepared in 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 0.02% C10E6. The enzyme and substrate solutions in individual syringes were mixed rapidly by triggering them into a mixing chamber driven through an optical cell and stopped with a third syringe. Different oxidation states of Fe-PPIX or Mn-PPIX were monitored spectroscopically. In the case of Fe3+-PPIX, the signal decay at 411 nm is due to the consumption of resting enzyme and the formation of Compound I. A peak shift to 420 nm represents the formation of Compound II and Intermediate II; these two species have the same UV-visible spectral features. There are two isosbestic points in the spectral scans; 427 nm between resting enzyme and Compound I and 410 nm between Compound I and Compound II-Intermediate II. The kinetic traces were extracted from spectra at isosbestic points and fitted to exponential equations to obtain pseudo-first-order rate constants (kobs). For the second-order reaction of Compound I formation, k1 was the slope from the linear part of a plot of pseudo-first-order rate constants k1(obs) versus hydroperoxide substrate concentrations. For Intermediate II formation, k2 was numerically equal to the maximum k2(obs) at saturating substrate concentrations for the intramolecular two-species reaction system. With ovPGHS-1 reconstituted with Mn3+-PPIX, the resting enzyme (3 μm) when mixed with 50 μm 15-HPETE showed decreases in absorbance at 372, 472, and 561 nm and a new peak at 417 nm. A consecutive three-species model (SpecFit, BioLogic Science Instruments) based on a singular value decomposition algorithm was exploited to resolve intermediates derived from Mn3+-PPIX ovPGHS-1. Kinetic traces were collected at 417 nm and fitted to two exponential equations. A three-exponential equation was applied for higher substrate concentrations when side reactions (e.g. reduction of oxidized heme intermediates or suicide inactivation) may be involved. Cyanide Binding—Purified ovPGHS-1 proteins (1–3 μm) were reconstituted with 0.8 mol of Fe3+-PPIX/protein monomer and incubated at room temperature for 30–60 min. The protein solution was centrifuged at 10,000 × g for 10 min, and 1 ml of the supernatant was transferred to a quartz cuvette. Aliquots of a concentrated NaCN stock solution were added to the protein solution to yield final cyanide concentrations of 0, 0.08, 0.16, 0.24, 0.32, 0.40, 0.65, 1.15, 1.65, 2.90, 4.15, 6.65, 11.65, 16.65, and 26.65 mm. The protein-ligand mixture was incubated for 5 min for each concentration of cyanide. The absorbance changes measured at 430 nm where the maximum change occurs were fitted to ΔAU =ΔAUmax × [CN–]/(Kd + [CN–]) to calculate a dissociation constant (Kd). Analysis of 15-HPETE Reaction Products—POX reactions were conducted in a 500-μl reaction mixture containing 50 nm native ovPGHS-1 or Q203V-ovPGHS-1 dimer, 50 nm hematin, and 4.5 mm phenol in 100 mm Tris-HCl, pH 8.0, at room temperature. The reactions were initiated by adding 15-HPETE (final concentration of 5 μm) and terminated at 2 min by adding 1.5 ml of a pre-cooled (4 °C) mixture of diethyl ether, methanol, 0.2 m citric acid (30:4:1). PPA (5 μm) was added as an internal control. The organic phase was shaken with 380 μl of a saturated NaCl solution at 4 °C to remove H2O. The upper organic layer was transferred to a clean tube and evaporated under N2. The products were dissolved in 100 μl of hexane:isopropanol: acetic acid (987:12:1; running solution) and resolved by HPLC on a Nucleosil Silica column (5 μm, 250 × 4.6 mm, PJ Cobert Associates) mounted on a Shimadzu HPLC system equipped with diode array detector. The bound products were eluted with running solution at a flow rate of 1 ml/min. 15-HPETE and 15-HETE were monitored at 235 nm, and 15-KETE was at 279 nm. PPA was eluted after the other standards; it has a peak absorbance at 249 nm but was monitored at 235 nm. HPLC tracings were converted to bitmap format, and peak areas were quantified using WinDIG 2.5 digitizing freeware. MP2-level Calculations of the Heterolytic Bond Dissociation of an Bond—The quantum chemical calculations were carried out by using the second-order Møller-Plesset perturbation method (MP2) as implemented in the Gaussian98 software package (37Frisch M.J. Trucks G.W. Schlegel H.B. Scuseria G.E. Robb M.A. Cheeseman J.R. Zakrzewski V.G. Montgomery Jr., J.A. Stratmann R.E. Burant J.C. Dapprich S. Millam J.M. Daniels A.D. Kudin K.N. Strain M.C. Farkas O. Tomasi J. Barone V. Cossi M. Cammi R. Mennucci B. Pomelli C. Adamo C. Clifford S. Ochterski J. Petersson G.A. Ayala P.Y. Cui Q. Morokuma K. Salvador P. Dannenberg J.J. Malick D.K. Rabuck A.D. Raghavachari K. Foresman J.B. Cioslowski J. Ortiz J.V. Baboul A.G. Stefanov B.B. Liu G. Liashenko A. Piskorz P. Komaromi I. Gomperts R. Martin R.L. Fox D.J. Keith T. Al-Laham M.A. Peng C.Y. Nanayakkara A. Challacombe M. Gill P.M.W. Johnson B. Chen W. Wong M.W. Andres J.L. Gonzalez C. Head-Gordon M. Replogle E.S. Pople J.A. Gaussian 98. Revision A.11.1 Ed. Gaussian, Inc., Pittsburg, PA2001Google Scholar). All the structures were geometry-optimized and their vibrational frequencies calculated using the 6–311++G** basis set in which polarization and diffuse functions are included. Molecular Dynamics (MD) Simulation of PGG2 Binding to the POX Site—The protocol for creating a model of PGG2, inserting it in ovPGHS-1, and running molecular dynamics on the complex is described in detail in our previous work on a similar substrate (29Seibold S.A. Smith W.L. Cukier R.I. J. Phys. Chem. B. 2004; 108: 9297-9305Crossref Scopus (5) Google Scholar). A brief summary is as follows. A molecular structure for PGG2 was generated using the MOE software package (38MOE Molecular Operating Environment. Chemical Computing Co., Montreal, Quebec H3B 3X3, Canada2003Google Scholar), and the resulting atom coordinates were optimized with use of Gaussian98 (37Frisch M.J. Trucks G.W. Schlegel H.B. Scuseria G.E. Robb M.A. Cheeseman J.R. Zakrzewski V.G. Montgomery Jr., J.A. Stratmann R.E. Burant J.C. Dapprich S. Millam J.M. Daniels A.D. Kudin K.N. Strain M.C. Farkas O. Tomasi J. Barone V. Cossi M. Cammi R. Mennucci B. Pomelli C. Adamo C. Clifford S. Ochterski J. Petersson G.A. Ayala P.Y. Cui Q. Morokuma K. Salvador P. Dannenberg J.J. Malick D.K. Rabuck A.D. Raghavachari K. Foresman J.B. Cioslowski J. Ortiz J.V. Baboul A.G. Stefanov B.B. Liu G. Liashenko A. Piskorz P. Komaromi I. Gomperts R. Martin R.L. Fox D.J. Keith T. Al-Laham M.A. Peng C.Y. Nanayakkara A. Challacombe M. Gill P.M.W. Johnson B. Chen W. Wong M.W. Andres J.L. Gonzalez C. Head-Gordon M. Replogle E.S. Pople J.A. Gaussian 98. Revision A.11.1 Ed. Gaussian, Inc., Pittsburg, PA2001Google Scholar). Atom-centered charges for the electrostatic part of the force field for MD were then generated with the Merz-Kollman method (37Frisch M.J. Trucks G.W. Schlegel H.B. Scuseria G.E. Robb M.A. Cheeseman J.R. Zakrzewski V.G. Montgomery Jr., J.A. Stratmann R.E. Burant J.C. Dapprich S. Millam J.M. Daniels A.D. Kudin K.N. Strain M.C. Farkas O. Tomasi J. Barone V. Cossi M. Cammi R. Mennucci B. Pomelli C. Adamo C. Clifford S. Ochterski J. Petersson G.A. Ayala P.Y. Cui Q. Morokuma K. Salvador P. Dannenberg J.J. Malick D.K. Rabuck A.D. Raghavachari K. Foresman J.B. Cioslowski J. Ortiz J.V. Baboul A.G. Stefanov B.B. Liu G. Liashenko A. Piskorz P. Komaromi I. Gomperts R. Martin R.L. Fox D.J. Keith T. Al-Laham M.A. Peng C.Y. Nanayakkara A. Challacombe M. Gill P.M.W. Johnson B. Chen W. Wong M.W. Andres J.L. Gonzalez C. Head-Gordon M. Replogle E.S. Pople J.A. Gaussian 98. Revision A.11.1 Ed. Gaussian, Inc., Pittsburg, PA2001Google Scholar). The coordinates of PGHS were obtained from the crystal structure (Protein Data Bank code 1CQE) of ovPGHS-1. The PGG2 structure was docked to PGHS by using a modified simulated annealing protocol, which found space for PGG2 by a fragment-based search procedure. The coordinates of the best-docked PGG2-PGHS complex, based on an energy criterion, were used to initiate a 1-ns MD simulation. The MD was carried out with the SANDER module of AMBER7 (39Case D.A. Pearlman D.A. Caldwell J.W. Wang J. Ross W.S. Simmerling C.L. Darden T.A. Mertz K.M. Stanton R.V. Cheng A.L. Vincent J.J. Crowley M. Tsue V. Gohlke H. Radmer R. Duan Y. Pitera J. Massova I. Seibel G.L. Singh C. Weiner P. Kollman P.A. AMBER Simulation Software Package, Version 7. Unversity of California, San Francisco, CA2002Google Scholar) using explicit solvent (∼5000 waters) in a 80 × 80 × 80-Å3 box, with the ionization states of all residues set appropriate to pH 7. Hydroperoxide Substrate Specificity of the POX Activity of ovPGHS-1—Previous studies have established that ovPGHS-1 catalyzes guaiacol oxidation efficiently with several primary and secondary alkyl hydroperoxide substrates (i.e. PGG2, 5-phenyl-4-pentenyl-1-hydroperoxide, and 15-HPETE (2Kulmacz R.J. van der Donk W.A. Tsai A.L. Prog. Lipid Res. 2003; 42: 377-404Crossref PubMed Scopus (75) Google Scholar, 3Rouzer C. Marnett L. Chem. Rev. 2003; 103: 2239-2304Crossref PubMed Scopus (195) Google Scholar, 24Lu G. Tsai A.L. Van Wart H.E. Kulmacz R.J. J. Biol. Chem. 1999; 274: 16162-16167Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 40DeGray J.A. Lassmann G. Curtis J.F. Kennedy T.A. Marnett L.J. Eling T.E. Mason R.P. J. Biol. Chem. 1992; 267: 23583-23588Abstract Full Text PDF PubMed Google Scholar). However, because the POX activity undergoes rapid suicide inactivation (35Song I. Ball" @default.
• W2082079362 created "2016-06-24" @default.
• W2082079362 creator A5025677580 @default.
• W2082079362 creator A5035388874 @default.
• W2082079362 creator A5042818934 @default.
• W2082079362 creator A5049645907 @default.
• W2082079362 creator A5072619513 @default.
• W2082079362 creator A5086549262 @default.
• W2082079362 date "2007-06-01" @default.
• W2082079362 modified "2023-10-15" @default.
• W2082079362 title "Prostaglandin Endoperoxide H Synthases" @default.
• W2082079362 cites W1490343139 @default.
• W2082079362 cites W1514181935 @default.
• W2082079362 cites W1529000927 @default.
• W2082079362 cites W1531513729 @default.
• W2082079362 cites W1536157072 @default.
• W2082079362 cites W1540849804 @default.
• W2082079362 cites W1556997704 @default.
• W2082079362 cites W1557310960 @default.
• W2082079362 cites W1592341453 @default.
• W2082079362 cites W1669371681 @default.
• W2082079362 cites W1722902827 @default.
• W2082079362 cites W1964786466 @default.
• W2082079362 cites W1968027839 @default.
• W2082079362 cites W1972433518 @default.
• W2082079362 cites W1972905772 @default.
• W2082079362 cites W1977602605 @default.
• W2082079362 cites W1978921937 @default.
• W2082079362 cites W1980189079 @default.
• W2082079362 cites W1980493203 @default.
• W2082079362 cites W1980742376 @default.
• W2082079362 cites W1980881765 @default.
• W2082079362 cites W1983725114 @default.
• W2082079362 cites W1983749281 @default.
• W2082079362 cites W1988578458 @default.
• W2082079362 cites W1993432693 @default.
• W2082079362 cites W1993938520 @default.
• W2082079362 cites W1995629951 @default.
• W2082079362 cites W2003092651 @default.
• W2082079362 cites W2004264939 @default.
• W2082079362 cites W2005709691 @default.
• W2082079362 cites W2005954426 @default.
• W2082079362 cites W2006133710 @default.
• W2082079362 cites W2007019274 @default.
• W2082079362 cites W2010967782 @default.
• W2082079362 cites W2011899140 @default.
• W2082079362 cites W2013777179 @default.
• W2082079362 cites W2014503546 @default.
• W2082079362 cites W2015399463 @default.
• W2082079362 cites W2016943233 @default.
• W2082079362 cites W2019328429 @default.
• W2082079362 cites W2021360223 @default.
• W2082079362 cites W2021687937 @default.
• W2082079362 cites W2023200971 @default.
• W2082079362 cites W2025670195 @default.
• W2082079362 cites W2027055405 @default.
• W2082079362 cites W2029941300 @default.
• W2082079362 cites W2030850720 @default.
• W2082079362 cites W2037094292 @default.
• W2082079362 cites W2038482404 @default.
• W2082079362 cites W2040981816 @default.
• W2082079362 cites W2042017985 @default.
• W2082079362 cites W2049551769 @default.
• W2082079362 cites W2049771771 @default.
• W2082079362 cites W2049959755 @default.
• W2082079362 cites W2050994990 @default.
• W2082079362 cites W2054508205 @default.
• W2082079362 cites W2054535723 @default.
• W2082079362 cites W2063408001 @default.
• W2082079362 cites W2068824753 @default.
• W2082079362 cites W2077315501 @default.
• W2082079362 cites W2078179845 @default.
• W2082079362 cites W2087561593 @default.
• W2082079362 cites W2090431078 @default.
• W2082079362 cites W2093683433 @default.
• W2082079362 cites W2093758951 @default.
• W2082079362 cites W2107277439 @default.
• W2082079362 cites W2108682052 @default.
• W2082079362 cites W2115350007 @default.
• W2082079362 cites W2126853519 @default.
• W2082079362 cites W2147929482 @default.
• W2082079362 cites W271818902 @default.
• W2082079362 cites W2894728782 @default.
• W2082079362 cites W2952210817 @default.
• W2082079362 doi "https://doi.org/10.1074/jbc.m701235200" @default.
• W2082079362 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17462992" @default.
• W2082079362 hasPublicationYear "2007" @default.
• W2082079362 type Work @default.
• W2082079362 sameAs 2082079362 @default.
• W2082079362 citedByCount "37" @default.
• W2082079362 countsByYear W20820793622012 @default.
• W2082079362 countsByYear W20820793622013 @default.
• W2082079362 countsByYear W20820793622014 @default.
• W2082079362 countsByYear W20820793622015 @default.
• W2082079362 countsByYear W20820793622016 @default.
• W2082079362 countsByYear W20820793622017 @default.
• W2082079362 countsByYear W20820793622018 @default.
• W2082079362 countsByYear W20820793622020 @default.

• next