Arachidonic Acid Oxygenation by COX-1 and COX-2

cyclooxygenase protein or the gene that codes for it non-steroidal anti-inflammatory drugs prostaglandin G2 Prostaglandins were discovered in human semen in 1930, but their low concentrations and instability precluded identification for nearly 30 years (for a brief historical review, see Ref. 1Bindra J.S. Bindra R. Prostaglandin Synthesis. Academic Press, New York1977: 7-22Crossref Google Scholar). Once they were identified, it was clear they arose from polyunsaturated fatty acids by a complex series of reactions involving oxygenation, cyclization, and the generation of five chiral centers from an achiral substrate. The mechanism of prostaglandin biosynthesis was outlined in 1967 by Hamberg and Samuelsson (2Hamberg M. Samuelsson B. J. Biol. Chem. 1967; 242: 5336-5343Abstract Full Text PDF PubMed Google Scholar), and the basic tenets have been confirmed in subsequent studies. The key step in their proposed mechanism was the formation of bicyclic peroxides (endoperoxides) as the initial products of polyunsaturated fatty acid oxygenation (Fig. 1). The term cyclooxygenase (COX)1 2The term cyclooxygenase is used to describe the enzyme activity or to refer to the active site for that activity on the protein.was coined to describe the enzyme that carried out this complex chemical transformation, and its role was confirmed by the isolation of prostaglandin endoperoxides in 1973 (3Hamberg M. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 899-903Crossref PubMed Scopus (570) Google Scholar, 4Nugteren D.H. Hazelhof E. Biochim. Biophys. Acta. 1973; 326: 448-461Crossref PubMed Scopus (635) Google Scholar). In addition to catalyzing a fascinating metabolic transformation, COX is an enormously important pharmacological target. Vane reported in 1971 (5Vane J.R. Nat. New Biol. 1971; 231: 232-235Crossref PubMed Scopus (7348) Google Scholar) that non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin formation and demonstrated that their relative inhibitory potency in vitro correlates to their anti-inflammatory activity in vivo . This not only explained the beneficial activity of NSAIDs but also their side effects such as gastrointestinal toxicity and bleeding because prostaglandins and related molecules (i.e. thromboxane) are involved in a very broad range of physiological and pathophysiological responses. The importance of these molecules as autocrine and paracrine mediators has been confirmed recently by the phenotypes of mice bearing targeted deletions inCOX genes or prostaglandin receptor genes. The discovery of a second gene (COX-2 ) coding for cyclooxygenase and the demonstration that its protein product is distributed differently from the originally discovered enzyme (COX-1) raised the possibility that some of the beneficial effects of NSAIDs may be separable from their side effects by development of isoform-selective inhibitors (6Fu J.-Y. Masferrer J.L. Seibert K. Raz A. Needleman P. J. Biol. Chem. 1990; 265: 16737-16740Abstract Full Text PDF PubMed Google Scholar, 7Xie W. Chipman J.G. Robertson D.L. Erikson R.L. Simmons D.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2692-2696Crossref PubMed Scopus (1686) Google Scholar, 8Kujubu D.A. Fletcher B.S. Varnum B.C. Lim R.W. Herschman H.R. J. Biol. Chem. 1991; 266: 12866-12872Abstract Full Text PDF PubMed Google Scholar, 9O'Banion M.K. Sadowski H.B. Winn V. Young D.A. J. Biol. Chem. 1991; 266: 23261-23267Abstract Full Text PDF PubMed Google Scholar). This hypothesis has been dramatically validated by the demonstration that selective COX-2 inhibitors are anti-inflammatory and analgesic but lack the gastric toxicity associated with all currently available NSAIDs (10Masferrer J.L. Zweifel B.S. Manning P.T. Hauser S.D. Leahy K.M. Smith W.G. Isakson P.C. Seibert K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3228-3232Crossref PubMed Scopus (1290) Google Scholar, 11Simon L.S. Lanza F.L. Lipsky P.E. Hubbard R.C. Talwalker S. Schwartz B.D. Isakson P.C. Geis G.S. Arthritis Rheum. 1998; 41: 1591-1602Crossref PubMed Scopus (490) Google Scholar). Substantial evidence supports the hypothesis that COX oxygenates arachidonic acid by a free radical mechanism (Fig. 1). Thus, COX appears to have co-opted the process that gives rise to isoprostanes to generate prostaglandins. The major differences between COX-catalyzed and spontaneous oxidation of arachidonic acid are the increased rate and high degree of stereochemical control of the enzymatic reaction (1 of 64 possible isomers predominates). Thus, the overall role of COX is rather simple: stereospecifically remove the 13-pro-S -hydrogen and control the stereochemistry of oxygenation. How does it do this? A protein tyrosyl radical appears to be the oxidizing agent that initiates arachidonic acid oxygenation (12Karthein R. Dietz R. Nastainczyk W. Ruf H.H. Eur. J. Biochem. 1988; 171: 313-320Crossref PubMed Scopus (231) Google Scholar). A tyrosyl radical is formed during COX turnover, and although there has been debate over the identity of the spectroscopically detected radicals, they appear capable of oxidizing arachidonic acid (13DeGray 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, 14Tsai A.-L. Palmer G. Kulmacz R.J. J. Biol. Chem. 1992; 267: 17753-17759Abstract Full Text PDF PubMed Google Scholar, 15Tsai A.-L. Palmer G. Xiao G. Swinney D.C. Kulmacz R.J. J. Biol. Chem. 1998; 273: 3888-3894Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The crystal structures of both COX-1 and COX-2 reveal that Tyr-385 is positioned perfectly to react with the fatty acid substrate (Fig.2) (16Picot D. Loll P.J. Garavito R.M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1151) Google Scholar, 17Luong C. Miller A. Barnett J. Chow J. Ramesha C. Browner M.F. Nat. Struct. Biol. 1996; 3: 927-933Crossref PubMed Scopus (561) Google Scholar, 18Kurumbail R.G. Stevens A.M. Gierse J.K. McDonald J.J. Stegeman R.A. Pak J.Y. Gildehaus D. Miyashiro J.M. Penning T.D. Seibert K. Isakson P.C. Stallings W.C. Nature. 1996; 384: 644-648Crossref PubMed Scopus (1600) Google Scholar). Indeed, the Y385F mutant is not catalytically active and does not oxidize arachidonic acid when it is treated with peroxide (19Shimokawa T. Kulmacz R.J. DeWitt D.L. Smith W.L. J. Biol. Chem. 1990; 265: 20073-20076Abstract Full Text PDF PubMed Google Scholar). Incubation of wild-type enzyme with arachidonate in the presence of nitric oxide quenches the EPR signal of the tyrosyl radical and leads to the formation of nitrotyrosine at position 385 in the protein (20Gunther M.R. Hsi L.C. Curtis J.F. Gierse J.K. Marnett L.J. Eling T.E. Mason R.P. J. Biol. Chem. 1997; 272: 17086-17090Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 21Goodwin D.C. Gunther M.H. Hsi L.H. Crews B.C. Eling T.E. Mason R.P. Marnett L.J. J. Biol. Chem. 1998; 273: 8903-8909Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Protein radicals require an oxidant for their formation, which in most cases is a metal-containing prosthetic group (22Stubbe J.S. van der Donk W.A. Chem. Rev. 1998; 98: 705-762Crossref PubMed Scopus (1364) Google Scholar). COX is a homodimer of 70-kDa subunits that each contain one molecule of heme (16Picot D. Loll P.J. Garavito R.M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1151) Google Scholar). The iron is ferric in the resting enzyme and is likely thermodynamically incapable of oxidizing Tyr-385 (E 12 = 0.9 V for Tyr⋅ → Tyr and E 12 = −0.2 to +0.2 V for Fe3+ → Fe2+ for most hemes) (22Stubbe J.S. van der Donk W.A. Chem. Rev. 1998; 98: 705-762Crossref PubMed Scopus (1364) Google Scholar, 23Kulmacz R.J. Ren Y. Tsai A.-L. Palmer G. Biochemistry. 1990; 29: 8760-8771Crossref PubMed Scopus (84) Google Scholar). Reaction of the heme of COX with peroxides generates a ferryl-oxo complex analogous to compound I of classic heme peroxidases (24Lambeir A.M. Markey C.M. Dunford H.B. Marnett L.J. J. Biol. Chem. 1985; 260: 14894-14896Abstract Full Text PDF PubMed Google Scholar). The redox potential of such higher oxidation states is typically on the order of +1 V so the compound I of COX is capable of oxidizing Tyr-385. Ruf and co-workers (25Dietz R. Nastainczyk W. Ruf H.H. Eur. J. Biochem. 1988; 171: 321-328Crossref PubMed Scopus (197) Google Scholar) demonstrated some time ago that oxidation of COX with organic hydroperoxides or fatty acid hydroperoxides generates a spectroscopically detectable tyrosyl radical, and they postulated that the tyrosyl radical oxidizes arachidonic acid. Support for this hypothesis is provided by the existence of significant lag phases for the induction of cyclooxygenase activity of Mn-protoporphyrin IX-reconstituted enzyme or site-directed mutants that exhibit diminished rates of reaction with hydroperoxide (26Smith W.L. Eling T.E. Kulmacz R.J. Marnett L.J. Tsai A. Biochemistry. 1992; 31: 3-7Crossref PubMed Scopus (132) Google Scholar). The identity of the hydroperoxide activator has been uncertain. Our laboratory has reported that peroxynitrite, the coupling product of nitric oxide and superoxide anion, is an excellent oxidant for the heme of COX and activates the enzyme even in the presence of concentrations of glutathione peroxidase and glutathione that inhibit activation by fatty acid hydroperoxides (27Landino L.M. Crews B.C. Timmons M.D. Morrow J.D. Marnett L.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15069-15074Crossref PubMed Scopus (398) Google Scholar). Activation is inhibited by superoxide dismutase, which scavenges peroxynitrite or prevents its formation from NO and O⨪2. Lipophilic superoxide dismutase mimetic agents inhibit prostaglandin biosynthesis by cultured mouse macrophages, which is consistent with a role for peroxynitrite in cyclooxygenase activation in intact cells. These findings provide a biochemical link between NO biosynthesis and prostaglandin biosynthesis and may explain the finding that NO synthase inhibitors reduce prostaglandin biosynthesis in inflammatory lesions in vivo (Equation 1) (28Salvemini D. Settle S.L. Masferrer J.L. Seibert K. Currie M.G. Needleman P. Br. J. Pharmacol. 1995; 114: 1171-1178Crossref PubMed Scopus (251) Google Scholar). Peroxynitrite activation of cyclooxygenase may be especially important in activated macrophages because inducible NO synthase and COX-2 are immediate early genes that are dramatically expressed in response to exposure to inflammatory stimuli such as lipopolysaccharide. The identity of the cyclooxygenase activator in non-inflammatory cells remains to be determined. There has been considerable experimental debate about whether the Tyr-385 radical is regenerated at the end of each cyclooxygenase catalytic cycle or requires reoxidation by another peroxidase catalytic cycle (29Bakovic M. Dunford H.B. Biochemistry. 1994; 33: 6475-6482Crossref PubMed Scopus (37) Google Scholar, 30Wei C. Kulmacz R.J. Tsai A.-L. Biochemistry. 1995; 34: 8499-8512Crossref PubMed Scopus (49) Google Scholar). Detailed kinetic investigations confirm observations extant at the onset of the debate that strongly support the regeneration of the catalytic tyrosyl radical at the end of each cycle of arachidonic acid oxidation (4Nugteren D.H. Hazelhof E. Biochim. Biophys. Acta. 1973; 326: 448-461Crossref PubMed Scopus (635) Google Scholar, 31Tsai A.-L. Wu G. Kulmacz R.J. Biochemistry. 1997; 36: 13085-13094Crossref PubMed Scopus (23) Google Scholar, 32Hamberg M. Svensson J. Wakabayashi T. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 345-349Crossref PubMed Scopus (872) Google Scholar). Thus, the final step in each round of arachidonic acid oxygenation is reduction of the peroxyl radical precursor of PGG2 by Tyr-385, which regenerates the Tyr-385 radical for the next round of cyclooxygenase catalysis (Fig.1). This leads to multiple turnovers per activation event and allows the accumulation of PGG2 (4Nugteren D.H. Hazelhof E. Biochim. Biophys. Acta. 1973; 326: 448-461Crossref PubMed Scopus (635) Google Scholar, 32Hamberg M. Svensson J. Wakabayashi T. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 345-349Crossref PubMed Scopus (872) Google Scholar). How does COX ensure that a single stereoisomer of PGG2 is produced from arachidonic acid? One can predict on purely chemical grounds that the enzyme must bind arachidonate in a conformation similar to that illustrated in Fig. 1. Removal of the 13-pro-S hydrogen followed by reaction with O2, serial cyclization, and reaction with the second O2 could occur with minimal motion of the reaction intermediates to produce PGG2 with all the correct stereocenters. We and others have docked arachidonate into the cyclooxygenase active site of sheep COX-1 to test whether such a conformation can be accommodated. The carboxylate was positioned adjacent to Arg-120, which plays a crucial role in binding arachidonate and arylalkanoic acid-type inhibitors (33Bhattacharyya D.K. Lecomte M. Rieke C.J. Garavito R.M. Smith W.L. J. Biol. Chem. 1996; 271: 2179-2184Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), and the 13-pro-S hydrogen was placed near the phenolic hydroxyl of Tyr-385. The ω-end of the fatty acid was inserted into a channel at the top of the cyclooxygenase active site that eventually leads to the surface of the protein. This end of arachidonate straddles the α-helix containing Ser-530, the site acetylated by aspirin. Acetylation by aspirin blocks arachidonate binding to COX-1 (34DeWitt D.L. El-Harith E.A. Kraemer S.A. Andrews M.J. Yao E.F. Armstrong R.L. Smith W.L. J. Biol. Chem. 1990; 265: 5192-5198Abstract Full Text PDF PubMed Google Scholar). The arachidonate-enzyme complex was then minimized to obtain the final conformation displayed in Fig.3. It is clear that the fatty acid substrate can be nicely accommodated within the active site in a conformation expected to yield PGG2. One prediction of this model is that O2 molecules diffuse up the central channel to couple to the solvent-exposed sides of the carbon radical intermediates to PGG2. This prediction is consistent with the that the 13-pro-S hydrogen is on the side of the fatty acid from the of both This model was for sheep but a similar model can be the for mouse The active site structures are similar for the of the with a that are this clear differences in substrate between the COX-2 appears COX-1 in that it oxidizes carbon polyunsaturated fatty acids with higher COX-2 oxidizes the of arachidonic acid COX-1 does not D.K. D.A. M. DeWitt D.L. Smith W.L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google M. D. Ramesha J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). with the the mutant of COX-1 a in for arachidonate the mutant of COX-2 for arachidonate that is similar to the wild-type enzyme (33Bhattacharyya D.K. Lecomte M. Rieke C.J. Garavito R.M. Smith W.L. J. Biol. Chem. 1996; 271: 2179-2184Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). C. A. R. and W. J. Biol. Chem. The for these differences is not between the is the of COX-2 to arachidonic acid to acid M. C. DeWitt D.L. Smith W.L. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, G. Tsai A.-L. Palmer G. W.C. P.J. Kulmacz R.J. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). COX-1 is to out this The importance of the channel at the top of the active site for binding the ω-end of arachidonate was recently confirmed by of at this position inhibit the oxidation of arachidonic acid but not fatty acids with at their S. B. C. and J. J. Biol. Chem. in The at the end of the a selective COX-2 a to of the have been been and are in L.J. Chem. Biol. 1998; PubMed Scopus Google Scholar, J.J. 1997; Scopus Google Scholar, P. D. Chem. 1997; Scopus Google Scholar, Chem. Biol. 1995; Full Text PDF PubMed Scopus Google Scholar). The most of inhibitors is of inhibitors and appear to be binding inhibitors in which the is in the second step (Equation R.A. J.M. J. S. M. D. S. J.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). This step the of the (E complex to the (E in which the is to the of the E in to and may the induction of a protein The is not associated with for most is the only COX that the protein Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). It Ser-530, which is to Arg-120, and it is COX-1 COX-2 (34DeWitt D.L. El-Harith E.A. Kraemer S.A. Andrews M.J. Yao E.F. Armstrong R.L. Smith W.L. J. Biol. Chem. 1990; 265: 5192-5198Abstract Full Text PDF PubMed Google Scholar, M. D.H. D.A. Eur. J. Biochem. PubMed Scopus Google Scholar). an molecule was that for COX-2 and only Crews B.C. C. Seibert K. Marnett L.J. 1998; PubMed Scopus Google Scholar, Crews B.C. Marnett L.J. J. Chem. 1998; 41: PubMed Scopus Google Scholar). structures of of sheep mouse and human COX-2 with and with selective inhibitors have been at (16Picot D. Loll P.J. Garavito R.M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1151) Google Scholar, 17Luong C. Miller A. Barnett J. Chow J. Ramesha C. Browner M.F. Nat. Struct. Biol. 1996; 3: 927-933Crossref PubMed Scopus (561) Google Scholar, 18Kurumbail R.G. Stevens A.M. Gierse J.K. McDonald J.J. Stegeman R.A. Pak J.Y. Gildehaus D. Miyashiro J.M. Penning T.D. Seibert K. Isakson P.C. Stallings W.C. Nature. 1996; 384: 644-648Crossref PubMed Scopus (1600) Google Scholar). NSAIDs with the group of Arg-120, which also with the carboxylate of arachidonate R.G. Stevens A.M. Gierse J.K. McDonald J.J. Stegeman R.A. Pak J.Y. Gildehaus D. Miyashiro J.M. Penning T.D. Seibert K. Isakson P.C. Stallings W.C. Nature. 1996; 384: 644-648Crossref PubMed Scopus (1600) Google Scholar, P.J. D. Garavito R.M. Nat. Struct. Biol. 1995; PubMed Scopus Google Scholar, P.J. D. Garavito R.M. Biochemistry. 1996; PubMed Scopus (170) Google Scholar). of the in COX-1 to or the protein to by NSAIDs (33Bhattacharyya D.K. Lecomte M. Rieke C.J. Garavito R.M. Smith W.L. J. Biol. Chem. 1996; 271: 2179-2184Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, J.A. D. P.J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). is of a with and which and the of the cyclooxygenase active site from the at the of the which the (Fig. (16Picot D. Loll P.J. Garavito R.M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1151) Google Scholar). of this the and binding and to occur C. Miller A. Barnett J. Chow J. Ramesha C. Browner M.F. Nat. Struct. Biol. 1996; 3: 927-933Crossref PubMed Scopus (561) Google Scholar). In the of the COX active which for the of arylalkanoic inhibitors the Swinney D.C. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). and of the may to the time of all COX of the (E complex in which the is to the especially bind to in COX-2 but not COX-1 is consistent with the hypothesis that these structures reveal the for their Fig. A that the in into a of COX-2 by R.G. Stevens A.M. Gierse J.K. McDonald J.J. Stegeman R.A. Pak J.Y. Gildehaus D. Miyashiro J.M. Penning T.D. Seibert K. Isakson P.C. Stallings W.C. Nature. 1996; 384: 644-648Crossref PubMed Scopus (1600) Google Scholar) and hydrogen to and the of R.G. Stevens A.M. Gierse J.K. McDonald J.J. Stegeman R.A. Pak J.Y. Gildehaus D. Miyashiro J.M. Penning T.D. Seibert K. Isakson P.C. Stallings W.C. Nature. 1996; 384: 644-648Crossref PubMed Scopus (1600) Google Scholar). A similar side the channel in COX-1 is not because of the presence of an of at position which between COX-2 and COX-1 that to of this side the and The COX-2 mutant is to by but not arylalkanoic acid-type NSAIDs J.K. McDonald J.J. Hauser S.D. C.M. Seibert K. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, E. C. D. J.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, L.H. Kulmacz R.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). the COX-1 mutant is to by E. C. D. J.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). of and of the or into the side may to the time of by The for by is on at the top rather the side of the cyclooxygenase active The of human COX-2 with a that the group is in van der with at the of the COX-2 active M. S. R. M. D. M. C. G. P. B. P. M. W. P. W. C. D. and Y. for in COX-1 is with which is not as by the group as at the top of the COX-1 active site may reduce the of the protein for the for its COX-2 all structures of COX-2 provide into the mechanism of The complex that the group to in a similar to NSAIDs rather into the side R. J.L. Stevens A.M. R.A. Gierse J.K. C.M. Seibert K. Isakson P.C. Stallings W.C. S. Marnett L.J. E. and in and New Scholar). the that with COX-2 is not from the of inhibitors does not provide a for their selective COX-2 The the at the of the COX active site and into the (Fig. ) C. Miller A. Barnett J. Chow J. Ramesha C. Browner M.F. Nat. Struct. Biol. 1996; 3: 927-933Crossref PubMed Scopus (561) Google Scholar). The of the hydrogen to Arg-120, and in COX-2 in a similar to the arylalkanoic acid these are to COX-1 and their importance in the of this for COX-2 is uncertain. The cyclooxygenase mechanism a of peroxidase and free radical The of protein radicals in arachidonate oxidation is but there is a of on the that generates there are of for tyrosyl radical generation that to be R.A. Penning Biochemistry. 1997; 36: PubMed Scopus Google Scholar). A of and is from and but the of these is be especially for in and binding is complex the model J. J. Marnett L.J. Biochemistry. 1998; PubMed Scopus Google Scholar). selective COX-2 inhibitors are on the and they are as in the as they have been in L.S. Lanza F.L. Lipsky P.E. Hubbard R.C. Talwalker S. Schwartz B.D. Isakson P.C. Geis G.S. Arthritis Rheum. 1998; 41: 1591-1602Crossref PubMed Scopus (490) Google Scholar), they a major by from bleeding is the potential selective inhibitors and COX mice have for this in a range of physiological and pathophysiological in human R.A. L.S. Lipsky P.E. J. 1998; PubMed Scopus Google Scholar). In it is likely that a range of structures of COX-2 inhibitors over the next years as by a of the for which have been recently is available at the The for and acids are and although this has not this remains an important and of the for the existence of COX genes expressed in the is not the of arachidonic acid for the remains and the mechanism by which inhibit COX-2 is not the to these not only provide important but also may for pharmacological a range of human We are to R. and M. Browner for and to B. and W. Smith for of and P. Isakson for with