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• W2034294731 abstract "The coupling between the peroxidase and cyclooxygenase activities of prostaglandin H synthase (PGHS) has been proposed to be mediated by a critical tyrosyl radical through a branched chain mechanism (Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 321-328). In this study, we have examined the ability of PGHS isoform-1 (PGHS-1) tyrosyl radicals to react with arachidonate. Anaerobic addition of arachidonate following formation of the peroxide-induced wide doublet or wide singlet tyrosyl radical led to disappearance of the tyrosyl radicals and emergence of a new EPR signal, which is distinct from known PGHS-1 tyrosyl radicals. The new radical was clearly derived from arachidonate because its EPR line shape changed when 5,6,8,9,11,12,14,15-octadeuterated arachidonate was used. Subsequent addition of oxygen to samples containing the fatty acyl radical resulted in regeneration of tyrosyl radical EPR. In contrast, the peroxide-generated tyrosyl radical in indomethacin-treated PGHS-1 (a narrow singlet) failed to react with arachidonate, consistent with the cyclooxygenase inhibition by indomethacin. These results indicate that the peroxide-generated wide doublet and wide singlet tyrosyl radicals serve as immediate oxidants of arachidonate bound at the cyclooxygenase active site to form a carbon-centered fatty acyl radical, which reacts with oxygen to form a hydroperoxide. These observations represent the first direct evidence of chemical coupling between the peroxidase reaction and arachidonate oxygenation in PGHS-1 and support the proposed role for a tyrosyl radical in cyclooxygenase catalysis. The coupling between the peroxidase and cyclooxygenase activities of prostaglandin H synthase (PGHS) has been proposed to be mediated by a critical tyrosyl radical through a branched chain mechanism (Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 321-328). In this study, we have examined the ability of PGHS isoform-1 (PGHS-1) tyrosyl radicals to react with arachidonate. Anaerobic addition of arachidonate following formation of the peroxide-induced wide doublet or wide singlet tyrosyl radical led to disappearance of the tyrosyl radicals and emergence of a new EPR signal, which is distinct from known PGHS-1 tyrosyl radicals. The new radical was clearly derived from arachidonate because its EPR line shape changed when 5,6,8,9,11,12,14,15-octadeuterated arachidonate was used. Subsequent addition of oxygen to samples containing the fatty acyl radical resulted in regeneration of tyrosyl radical EPR. In contrast, the peroxide-generated tyrosyl radical in indomethacin-treated PGHS-1 (a narrow singlet) failed to react with arachidonate, consistent with the cyclooxygenase inhibition by indomethacin. These results indicate that the peroxide-generated wide doublet and wide singlet tyrosyl radicals serve as immediate oxidants of arachidonate bound at the cyclooxygenase active site to form a carbon-centered fatty acyl radical, which reacts with oxygen to form a hydroperoxide. These observations represent the first direct evidence of chemical coupling between the peroxidase reaction and arachidonate oxygenation in PGHS-1 and support the proposed role for a tyrosyl radical in cyclooxygenase catalysis. INTRODUCTIONProstaglandin H synthase (PGHS)1( 1The abbreviations used are: PGHS-1prostaglandin H synthase isoform-1PGG2prostaglandin G2PGH2prostaglandin H2d8-arachidonate5,6,8,9,11,12,14,15-octadeuterated arachidonateWDwide doublet tyrosyl radicalWSwide singlet tyrosyl radicalNSnarrow singlet tyrosyl radical. ) plays a key role in controlling the biosynthesis of various physiologically important prostaglandins (1Samuelsson B. Goldyne M. Granström E. Hamberg M. Hammarström S. Malmsten C. Annu. Rev. Biochem. 1978; 47: 997-1029Crossref PubMed Scopus (972) Google Scholar). Although two distinct PGHS isozymes have been discovered in many different cells or tissues, they are believed to share the same basic catalytic mechanism (2Smith W.L. Eling T.E. Kulmacz R.J. Marnett L.J. Tsai A.-L. Biochemistry. 1992; 31: 3-7Crossref PubMed Scopus (132) Google Scholar). Type 1 PGHS (PGHS-1) thus serves as a useful prototype for detailed mechanistic study. PGHS-1 has two enzyme functions: a cyclooxygenase, which converts arachidonic acid to PGG2, and a peroxidase, which catalyzes the transformation of PGG2 to PGH2(1Samuelsson B. Goldyne M. Granström E. Hamberg M. Hammarström S. Malmsten C. Annu. Rev. Biochem. 1978; 47: 997-1029Crossref PubMed Scopus (972) Google Scholar, 2Smith W.L. Eling T.E. Kulmacz R.J. Marnett L.J. Tsai A.-L. Biochemistry. 1992; 31: 3-7Crossref PubMed Scopus (132) Google Scholar, 3Miyamoto T. Ogino N. Yamamoto S. Hayaishi O. J. Biol. Chem. 1976; 251: 2629-2636Abstract Full Text PDF PubMed Google Scholar). A branched chain radical mechanism (Fig. SI) has been proposed to integrate the two catalytic activities (4Dietz R. Nastainczyk W. Ruf H.H. Eur. J. Biochem. 1988; 171: 321-328Crossref PubMed Scopus (194) Google Scholar). In this proposal, a tyrosyl radical (Fe(IV)Tyr) is generated by an internal electron transfer in peroxidase compound I (Fe(V)), shown as step 2. The cyclooxygenase reaction itself begins when the tyrosyl radical reacts with bound arachidonate (AA) to form a fatty acyl radical (Fe(IV)Tyr/AA), shown as step 3. The bound fatty acyl radical then reacts with molecular oxygen and rearranges to form a PGG2 radical (step 4). In the final cyclooxygenase step, the tyrosyl radical is regenerated, and PGG2 is released (step 5).The tyrosyl radical mechanism nicely explains the heme dependence of both enzyme activities and the hydroperoxide requirement of the cyclooxygenase activity, and it also is consistent with x-ray crystallographic data that interposes a tyrosine residue (Tyr-385) between the heme and the arachidonic acid binding channel (5Picot D. Loll P.J. Garavito R.M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1141) Google Scholar). Results from chemical modification and site-directed mutagenesis also have suggested a critical role for Tyr-385 in catalysis and radical formation (6Shimokawa T. Kulmacz R.J. DeWitt D.L. Smith W.L. J. Biol. Chem. 1990; 265: 20073-20076Abstract Full Text PDF PubMed Google Scholar, 7Tsai A. Hsi L.C. Kulmacz R.J. Palmer G. Smith W.L. J. Biol. Chem. 1994; 269: 5085-5091Abstract Full Text PDF PubMed Google Scholar). Further, the kinetic correlation of cyclooxygenase product formation and tyrosyl radical levels has provided indirect evidence that a specific wide doublet tyrosyl radical (WD) could serve as the immediate oxidizing agent in the cyclooxygenase cycle (8Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar). However, other PGHS-1 tyrosyl radical species also are observed (8Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar, 9Kulmacz R.J. Ren Y. Tsai A.-L. Palmer G. Biochemistry. 1990; 29: 8760-8771Crossref PubMed Scopus (84) Google Scholar, 10Lassmann G. Odenwaller R. Curtis J.F. DeGray J.A. Mason R.P. Marnett L.J. Eling T.E. J. Biol. Chem. 1991; 266: 20045-20055Abstract Full Text PDF PubMed Google Scholar, 11DeGray 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), and direct evidence for reaction of any of the radicals with arachidonate has been lacking. In the studies described here, we have used single turnover experiments to demonstrate that the peroxidase-associated WD radical indeed reacts with arachidonic acid to form a carbon-centered radical, providing direct evidence for a key step in the proposed tyrosyl radical mechanism.MATERIALS AND METHODSArachidonic acid was purchased from NuChek Preps, Inc. (Elysian, MN). 5,6,8,9,11,12,14,15-Octadeuterated arachidonic acid was a generous gift of Hoffman-La Roche (Nutley, NJ). Polar contaminants were not detected in either deuterated or unlabeled arachidonic acid when analyzed by silica gel thin layer chromatography with hexane/diethyl ether/acetic acid (60:60:1) as the solvent. Mass spectral analysis of the methyl ester of the deuterated arachidonate indicated a composition of 76% octadeuteration and 24% heptadeuteration. The deuterated arachidonate was not purified further. Hemin and indomethacin were obtained from Sigma. Ethyl hydroperoxide was the product of Polyscience Inc. (Warrington, PA).PGHS-1 was purified from sheep seminal vesicles (12Kulmacz R.J. Lands W.E.M. Benedetto C. McDonald-Gibson R.G. Nigam S. Slater R.F. Prostaglandins and Related Substances: A Practical Approach. IRL Press, Washington D. C.1987: 209-227Google Scholar). The holoenzyme was prepared by replenishing the lost heme as previously described (8Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar). Cyclooxygenase activity was assayed by oxygen consumption rate (12Kulmacz R.J. Lands W.E.M. Benedetto C. McDonald-Gibson R.G. Nigam S. Slater R.F. Prostaglandins and Related Substances: A Practical Approach. IRL Press, Washington D. C.1987: 209-227Google Scholar); enzyme preparations used in this study had specific activities ranging from 65 to 80 μmol of O2/min/mg of protein.The glass titration vessel used in our single turnover experiments (Fig. SII) was adapted from the original design of Dutton (13Dutton P.L. Biochim. Biophys. Acta. 1971; 226: 63-81Crossref PubMed Scopus (347) Google Scholar, 14Tsai A.-L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (53) Google Scholar). The PGHS-1 enzyme solution (1.1-1.5 ml) was placed in the glass vessel, and the opening was sealed by clamping down the hard plastic cap lined with a rubber gasket. The bottom portion of the glass vessel was immersed in ice water, and the enzyme was constantly stirred with a glass-encased magnet to keep the enzyme solution at 0°C. The vessel and contents were made anaerobic by five cycles of alternating vacuum (30 s) and argon replacement (5 min) through a glass valve connected to an anaerobic train (14Tsai A.-L. Palmer G. Biochim. Biophys. Acta. 1983; 722: 349-363Crossref PubMed Scopus (53) Google Scholar). Arachidonic acid and ethyl hydroperoxide solutions contained in glass tubes sealed with rubber septa were made anaerobic by bubbling with argon for 5 min on ice. The substrates were then added in the desired sequence through the rubber septum plugs on the titration vessel cap using gas-tight Hamilton syringes.Scheme II:Diagram of the anaerobic vessel used. 1, gas-tight Hamilton syringes; 2, butyl rubber tubing connecting to the anaerobic train; 3, stainless steel transfer needle (wrapped to minimize warming from the fingers during manipulation); 4, hollow glass high-vacuum stopcock; 5, quartz EPR tube; 6, rubber septa; 7, hard plastic cap; 8, metal clamp; 9, rubber gasket; 10, glass vessel; 11, well for enzyme solution; 12, stirring magnet.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Aliquots of the reaction mixtures were transferred to EPR tubes through a long stainless steel transfer needle using the positive argon pressure in the vessel. For this, the transfer needle was first positioned in the titrator headspace, and the argon venting from the outside end of the needle was used to flush a prechilled EPR tube for a few seconds. Then, the tip of the needle was lowered into the enzyme solution to begin transfer. After the EPR tube was filled to a depth of about 3 cm, transfer was completed by raising the tip of the needle above the liquid surface. While still being flushed with argon, the EPR sample was frozen quickly in a dry ice/acetone bath and stored in liquid nitrogen for EPR analysis. Further aerobic reaction of individual EPR samples was achieved by immersion in a 0°C bath and mixing with a nichrome wire plunger for the desired time interval before refreezing the sample in the dry ice/acetone bath.EPR spectra were recorded at liquid nitrogen or liquid helium temperatures on a Varian E-6 spectrometer (8Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar). Unless otherwise noted, the EPR conditions for liquid nitrogen temperature were as follows: modulation amplitude, 2 G; time constant, 1 s; power, 1 milliwatt; and temperature, 96 K. Radical concentrations were determined by double integration of the EPR signals, with reference to a copper standard (15Wertz J.E. Bolton J.R. Electron Spin Resonance. Chapman and Hall, New York1986: 462-464Google Scholar). Computer simulations of EPR spectra were done on a PC using a modified version of the POWFUN program (16Hoganson C.W. Babcock G.T. Biochemistry. 1992; 31: 11874-11880Crossref PubMed Scopus (138) Google Scholar) kindly provided by Dr. Gerald T. Babcock and Dr. Curt Hoganson (Michigan State University).DISCUSSIONA key step in the mechanism in Fig. SI is the reaction of the hydroperoxide-induced tyrosyl radical with bound arachidonate to activate the fatty acid for reaction with oxygen. The reduction potential of the tyrosyl radical (TyrO/TyrOH) in solution at pH 7 is 0.94 V versus NHE (19DeFelippis M.R. Murthy C.P. Faraggi M. Klapper M.H. Biochemistry. 1989; 28: 4847-4853Crossref PubMed Scopus (185) Google Scholar). Abstraction of hydrogen from the fatty acid gives a pentadienyl radical; this radical has a reduction potential (R/RH) of 0.60 V (20Koppenol W.H. FEBS Lett. 1990; 264: 165-167Crossref PubMed Scopus (128) Google Scholar). A tyrosyl radical is thus a strong enough oxidant to abstract a hydrogen atom from arachidonate. The tyrosine residue believed to carry the unpaired electron in native PGHS-1, Tyr-385 (6Shimokawa T. Kulmacz R.J. DeWitt D.L. Smith W.L. J. Biol. Chem. 1990; 265: 20073-20076Abstract Full Text PDF PubMed Google Scholar, 7Tsai A. Hsi L.C. Kulmacz R.J. Palmer G. Smith W.L. J. Biol. Chem. 1994; 269: 5085-5091Abstract Full Text PDF PubMed Google Scholar), is located in the vicinity of both the heme and the putative fatty acid binding site (5Picot D. Loll P.J. Garavito R.M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1141) Google Scholar). Thus, having a tyrosyl radical in PGHS-1 as the oxidant for bound arachidonate is quite plausible from the structural standpoint. The new radical produced by reaction of arachidonate with tyrosyl radical is established as a fatty acyl radical by the dramatic change in line shape observed when deuterated arachidonate was used (Figure 2:, Figure 3:, Figure 4:). This demonstration of the ability of tyrosyl radical to form fatty acyl radical provides the first direct evidence that tyrosyl radical in native PGHS-1 actually does react with arachidonic acid as required by the proposed mechanism (Fig. SI) and is not just a marker for self-inactivation as suggested by other investigators (10Lassmann G. Odenwaller R. Curtis J.F. DeGray J.A. Mason R.P. Marnett L.J. Eling T.E. J. Biol. Chem. 1991; 266: 20045-20055Abstract Full Text PDF PubMed Google Scholar, 11DeGray 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).Given the arrangement of double bonds in arachidonate and the fact that the fatty acid-derived radical was trapped in the absence of oxygen, this radical is most likely a pentadienyl carbon-centered radical, as depicted in Fig. 7. The neutral radical formed from abstraction of the 13-pro- S hydrogen atom of arachidonate would be expected to have its electron density delocalized over C-11-C-15, as found in model systems containing the pentadienyl structure (21Sustmann R. Schmidt H. Chem. Ber. 1979; 112: 1440-1447Crossref Scopus (36) Google Scholar, 22Hinchliffe A. Cobb J. J. Mol. Struct. 1974; 23: 273-279Crossref Scopus (9) Google Scholar, 23Bascetta E. Gunstone F.D. Scrimgeour C.M. Walton J.C. J. Chem. Soc. Chem. Commun. 1982; : 110-112Crossref Google Scholar, 24Davies A.G. Griller D. Ingold K.U. Lindsay D.A. Walton J.C. J. Chem. Soc. Perkin Trans. 1981; 2: 633-641Crossref Scopus (65) Google Scholar) and in the substrate-associated purple lipoxygenase radical (25Nelson M.J. Seitz S.P. Cowling R.A. Biochemistry. 1990; 29: 6897-6903Crossref PubMed Scopus (76) Google Scholar, 26Nelson M.J. Cowling R.A. Seitz S.P. Biochemistry. 1994; 33: 4966-4973Crossref PubMed Scopus (80) Google Scholar). The unpaired electron density is likely to be higher at C-11, C-13, and C-15 and lower at C-12 and C-14 (21Sustmann R. Schmidt H. Chem. Ber. 1979; 112: 1440-1447Crossref Scopus (36) Google Scholar, 22Hinchliffe A. Cobb J. J. Mol. Struct. 1974; 23: 273-279Crossref Scopus (9) Google Scholar, 23Bascetta E. Gunstone F.D. Scrimgeour C.M. Walton J.C. J. Chem. Soc. Chem. Commun. 1982; : 110-112Crossref Google Scholar, 24Davies A.G. Griller D. Ingold K.U. Lindsay D.A. Walton J.C. J. Chem. Soc. Perkin Trans. 1981; 2: 633-641Crossref Scopus (65) Google Scholar). Spectral simulations were performed using the proton hyperfine coupling constants observed for a planar pentadienyl radical (21Sustmann R. Schmidt H. Chem. Ber. 1979; 112: 1440-1447Crossref Scopus (36) Google Scholar, 22Hinchliffe A. Cobb J. J. Mol. Struct. 1974; 23: 273-279Crossref Scopus (9) Google Scholar, 23Bascetta E. Gunstone F.D. Scrimgeour C.M. Walton J.C. J. Chem. Soc. Chem. Commun. 1982; : 110-112Crossref Google Scholar, 24Davies A.G. Griller D. Ingold K.U. Lindsay D.A. Walton J.C. J. Chem. Soc. Perkin Trans. 1981; 2: 633-641Crossref Scopus (65) Google Scholar) as initial values and a range of isotropic coupling constants for the β-protons. It was assumed that the principal axes of the A and g tensors are parallel and that only one out of the two β-protons at C-10 and C-16 has significant interaction with the unpaired electron. The optimal spectrum fits achieved for the radicals from unlabeled and deuterated arachidonate are shown together with the original EPR spectra in Fig. 8. The values for the parameters used in the simulations are summarized in Table I, which also includes data from a model planar pentadienyl radical.Figure 7:Proposed structures of planar pentadienyl radicals generated by abstraction of the 13-pro(S) hydrogen from unlabeled arachidonate (upper) or d8-arachidonate (lower).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8:Simulations of EPR spectra of radicals derived from unlabeled arachidonate (A) and from d8-arachidonate (B). The simulated spectra (dottedlines) were calculated as described in the text and are compared with the actual spectra (solidlines).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IParameters used to simulate EPR spectra of radicals observed in reactions with unlabeled and deuterated arachidonate Open table in a new tab A satisfactory simulation was obtained for the radical derived from unlabeled arachidonic acid (Fig. 8 A and Table I) using coupling constants of 1 (2H), 9.8 (2H), 5 (1H), 7 (1H), and 12 (1H) G. A line width of 4.1 G was used to reflect the poor resolution in the original spectrum. The hyperfine coupling constants for the first two sets of doublet protons can thus be assigned to those associated with C-12/C-14 and C-11/C-15, respectively, whereas the other three coupling constants are associated with the protons at C-13 and one each of the β-protons associated with either C-10 or C-16 (Fig. 7). The EPR spectrum produced by reaction of PGHS-1 tyrosyl radical with arachidonate can thus be reasonably ascribed to the expected carbon-centered pentadienyl radical on the fatty acid. The radical observed directly in the present experiments may well be the parent of a spin-trapped adduct previously reported (27Schreiber J. Eling T.E. Mason R.P. Arch. Biochem. Biophys. 1986; 249: 126-136Crossref PubMed Scopus (58) Google Scholar).The simulation for the EPR signal with deuterated arachidonate was less satisfactory (Fig. 8 B). Based on the chemical structure shown in Fig. 7, the radical spectrum would be expected to have proton hyperfine structure caused by three protons: the single proton at C-13 and the β-proton pairs at C-10 and C-16. Such splitting would produce a quadruplet radical EPR. However, a fifth splitting was actually observed in samples prepared from three different batches of PGHS-1 (Fig. 8 B). This additional splitting may reflect the additional proton in the 24 mol % of d7-arachidonate present in the deuterated arachidonate used for the experiments. However, the location(s) of the additional proton could not be determined from the mass spectral analyses, and so no attempt was made to account for it explicitly in the EPR simulation, which was based on d8-arachidonate. The coupling constants giving the optimal simulation for the d8-arachidonate radical structure (Figs. 7 and 8 and Table I) are 1 (4H), 9.6 (1H), 11.6 (1H), and 13 (1H) G and a line width of 5 G. As the hyperfine interaction from a deuteron is only one-sixth that of a proton, the first four equivalent protons thus are the deuterons substituted at C-11, C-12, C-14, and C-15 (Fig. 7).A tyrosyl radical plays a central role in the branched chain mechanism proposed for cyclooxygenase catalysis by PGHS-1 (Fig. SI and Ref. 4Dietz R. Nastainczyk W. Ruf H.H. Eur. J. Biochem. 1988; 171: 321-328Crossref PubMed Scopus (194) Google Scholar). As required by the mechanism, reaction of pure PGHS-1 with hydroperoxide can produce a tyrosyl radical (9Kulmacz R.J. Ren Y. Tsai A.-L. Palmer G. Biochemistry. 1990; 29: 8760-8771Crossref PubMed Scopus (84) Google Scholar, 28Karthein R. Dietz R. Nastainczyk W. Ruf H.H. Eur. J. Biochem. 1988; 171: 313-320Crossref PubMed Scopus (230) Google Scholar). In fact, detailed EPR studies have detected at least three types of hydroperoxide-induced radical signals in the enzyme, all ascribed to tyrosyl radicals (8Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar, 9Kulmacz R.J. Ren Y. Tsai A.-L. Palmer G. Biochemistry. 1990; 29: 8760-8771Crossref PubMed Scopus (84) Google Scholar, 10Lassmann G. Odenwaller R. Curtis J.F. DeGray J.A. Mason R.P. Marnett L.J. Eling T.E. J. Biol. Chem. 1991; 266: 20045-20055Abstract Full Text PDF PubMed Google Scholar, 28Karthein R. Dietz R. Nastainczyk W. Ruf H.H. Eur. J. Biochem. 1988; 171: 313-320Crossref PubMed Scopus (230) Google Scholar). A WD arising from a tyrosyl radical with a strained ring orientation is observed early during reaction of native enzyme with hydroperoxide (9Kulmacz R.J. Ren Y. Tsai A.-L. Palmer G. Biochemistry. 1990; 29: 8760-8771Crossref PubMed Scopus (84) Google Scholar, 17Barry B.A. El-Deeb M.K. Sandusky P.O. Babcock G.T. J. Biol. Chem. 1990; 265: 20139-20143Abstract Full Text PDF PubMed Google Scholar, 28Karthein R. Dietz R. Nastainczyk W. Ruf H.H. Eur. J. Biochem. 1988; 171: 313-320Crossref PubMed Scopus (230) Google Scholar). PGHS-1, whose cyclooxygenase activity has been inhibited with agents such as indomethacin, produces NS EPR characteristic of a tyrosyl radical with a relaxed ring orientation (8Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar, 9Kulmacz R.J. Ren Y. Tsai A.-L. Palmer G. Biochemistry. 1990; 29: 8760-8771Crossref PubMed Scopus (84) Google Scholar, 17Barry B.A. El-Deeb M.K. Sandusky P.O. Babcock G.T. J. Biol. Chem. 1990; 265: 20139-20143Abstract Full Text PDF PubMed Google Scholar). PGHS-1 inactivated during catalysis also produces an NS EPR but with somewhat less distinct hyperfine features than observed with the indomethacin-treated enzyme (8Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar, 9Kulmacz R.J. Ren Y. Tsai A.-L. Palmer G. Biochemistry. 1990; 29: 8760-8771Crossref PubMed Scopus (84) Google Scholar). WS EPR signals also are observed (8Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar, 9Kulmacz R.J. Ren Y. Tsai A.-L. Palmer G. Biochemistry. 1990; 29: 8760-8771Crossref PubMed Scopus (84) Google Scholar, 10Lassmann G. Odenwaller R. Curtis J.F. DeGray J.A. Mason R.P. Marnett L.J. Eling T.E. J. Biol. Chem. 1991; 266: 20045-20055Abstract Full Text PDF PubMed Google Scholar, 11DeGray 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). The EPR of the WS radical produced under self-inactivation conditions can be accounted for by the sum of the corresponding WD and NS radicals (10Lassmann G. Odenwaller R. Curtis J.F. DeGray J.A. Mason R.P. Marnett L.J. Eling T.E. J. Biol. Chem. 1991; 266: 20045-20055Abstract Full Text PDF PubMed Google Scholar, 11DeGray 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) but not by the sum of the WD from native enzyme and the NS from indomethacin-treated enzyme.2( 2A.-L. Tsai, unpublished results. ) Thus, there may actually be two types of NS radicals. Comparisons of the kinetics of the various tyrosyl radicals with the cyclooxygenase kinetics have indicated that the WD radical is present at appreciable concentrations during cyclooxygenase catalysis (8Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar).It is important to note that the WD and WS radicals associated with PGHS-1 with active cyclooxygenase (8Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar) reacted with arachidonate (Figure 2:, Figure 3:, Figure 4:), whereas the NS radical associated with inactivated cyclooxygenase (9Kulmacz R.J. Ren Y. Tsai A.-L. Palmer G. Biochemistry. 1990; 29: 8760-8771Crossref PubMed Scopus (84) Google Scholar) did not react with the fatty acid (Fig. 6). This demonstrates that reaction with arachidonate requires particular PGHS-1 tyrosyl radicals, and it links the reaction to cyclooxygenase catalysis rather than to nonspecific fatty acid oxidation. Further, the observation that the fatty acyl radical is converted back to the tyrosyl radical upon reaction with oxygen is exactly the behavior proposed for the arachidonate radical intermediate in cyclooxygenase catalysis (Fig. SI). The evidence placing the reaction of tyrosyl radical with arachidonate within the context of cyclooxygenase catalysis indicates that the observed reaction corresponds to step 3 in the mechanism in Fig. SI. In summary, the results of the present study provide strong support for the key initial cyclooxygenase step in the tyrosyl radical mechanism proposed for PGHS-1 catalysis. INTRODUCTIONProstaglandin H synthase (PGHS)1( 1The abbreviations used are: PGHS-1prostaglandin H synthase isoform-1PGG2prostaglandin G2PGH2prostaglandin H2d8-arachidonate5,6,8,9,11,12,14,15-octadeuterated arachidonateWDwide doublet tyrosyl radicalWSwide singlet tyrosyl radicalNSnarrow singlet tyrosyl radical. ) plays a key role in controlling the biosynthesis of various physiologically important prostaglandins (1Samuelsson B. Goldyne M. Granström E. Hamberg M. Hammarström S. Malmsten C. Annu. Rev. Biochem. 1978; 47: 997-1029Crossref PubMed Scopus (972) Google Scholar). Although two distinct PGHS isozymes have been discovered in many different cells or tissues, they are believed to share the same basic catalytic mechanism (2Smith W.L. Eling T.E. Kulmacz R.J. Marnett L.J. Tsai A.-L. Biochemistry. 1992; 31: 3-7Crossref PubMed Scopus (132) Google Scholar). Type 1 PGHS (PGHS-1) thus serves as a useful prototype for detailed mechanistic study. PGHS-1 has two enzyme functions: a cyclooxygenase, which converts arachidonic acid to PGG2, and a peroxidase, which catalyzes the transformation of PGG2 to PGH2(1Samuelsson B. Goldyne M. Granström E. Hamberg M. Hammarström S. Malmsten C. Annu. Rev. Biochem. 1978; 47: 997-1029Crossref PubMed Scopus (972) Google Scholar, 2Smith W.L. Eling T.E. Kulmacz R.J. Marnett L.J. Tsai A.-L. Biochemistry. 1992; 31: 3-7Crossref PubMed Scopus (132) Google Scholar, 3Miyamoto T. Ogino N. Yamamoto S. Hayaishi O. J. Biol. Chem. 1976; 251: 2629-2636Abstract Full Text PDF PubMed Google Scholar). A branched chain radical mechanism (Fig. SI) has been proposed to integrate the two catalytic activities (4Dietz R. Nastainczyk W. Ruf H.H. Eur. J. Biochem. 1988; 171: 321-328Crossref PubMed Scopus (194) Google Scholar). In this proposal, a tyrosyl radical (Fe(IV)Tyr) is generated by an internal electron transfer in peroxidase compound I (Fe(V)), shown as step 2. The cyclooxygenase reaction itself begins when the tyrosyl radical reacts with bound arachidonate (AA) to form a fatty acyl radical (Fe(IV)Tyr/AA), shown as step 3. The bound fatty acyl radical then reacts with molecular oxygen and rearranges to form a PGG2 radical (step 4). In the final cyclooxygenase step, the tyrosyl radical is regenerated, and PGG2 is released (step 5).The tyrosyl radical mechanism nicely explains the heme dependence of both enzyme activities and the hydroperoxide requirement of the cyclooxygenase activity, and it also is consistent with x-ray crystallographic data that interposes a tyrosine residue (Tyr-385) between the heme and the arachidonic acid binding channel (5Picot D. Loll P.J. Garavito R.M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1141) Google Scholar). Results from chemical modification and site-directed mutagenesis also have suggested a critical role for Tyr-385 in catalysis and radical formation (6Shimokawa T. Kulmacz R.J. DeWitt D.L. Smith W.L. J. Biol. Chem. 1990; 265: 20073-20076Abstract Full Text PDF PubMed Google Scholar, 7Tsai A. Hsi L.C. Kulmacz R.J. Palmer G. Smith W.L. J. Biol. Chem. 1994; 269: 5085-5091Abstract Full Text PDF PubMed Google Scholar). Further, the kinetic correlation of cyclooxygenase product formation and tyrosyl radical levels has provided indirect evidence that a specific wide doublet tyrosyl radical (WD) could serve as the immediate oxidizing agent in the cyclooxygenase cycle (8Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar). However, other PGHS-1 tyrosyl radical species also are observed (8Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar, 9Kulmacz R.J. Ren Y. Tsai A.-L. Palmer G. Biochemistry. 1990; 29: 8760-8771Crossref PubMed Scopus (84) Google Scholar, 10Lassmann G. Odenwaller R. Curtis J.F. DeGray J.A. Mason R.P. Marnett L.J. Eling T.E. J. Biol. Chem. 1991; 266: 20045-20055Abstract Full Text PDF PubMed Google Scholar, 11DeGray 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), and direct evidence for reaction of any of the radicals with arachidonate has been lacking. In the studies described here, we have used single turnover experiments to demonstrate that the peroxidase-associated WD radical indeed reacts with arachidonic acid to form a carbon-centered radical, providing direct evidence for a key step in the proposed tyrosyl radical mechanism." @default.
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• W2034294731 title "Spectroscopic Evidence for Reaction of Prostaglandin H Synthase-1 Tyrosyl Radical with Arachidonic Acid" @default.
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