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The enzymology of prostaglandin endoperoxide H synthases-1 and -2
Prostaglandins & other Lipid Mediators 68–69 (2002) 115–128
The enzymology of prostaglandin endoperoxide H synthases-1 and -2 William L. Smith a,∗ , Inseok Song b a
Department of Biochemistry and Molecular Biology, Michigan State University, 513 Biochemistry Building, East Lansing, MI 48824 USA b Department of Life Science, University of Seoul, Seoul, South Korea
Abstract We summarize the enzymological properties of prostaglandin endoperoxide H synthases (PGHs)-1 and -2, the enzymes that catalyze the committed step in prostaglandin biosynthesis. These isoenzymes are closely related structurally and mechanistically. Each catalyzes a peroxidase and a cyclooxygenase reaction at spatially separate but neighboring, electronically interrelated active sites. The peroxidase is necessary to activate the cyclooxygenase; oxidation of the heme group of the peroxidase by peroxide leads to oxidation of a cyclooxygenase active site tyrosine. The tyrosine radical abstracts hydrogen from arachidonic acid to form an arachidonate radical which reacts sequentially with two oxygen molecules forming the intermediate product PGG2. PGG2 is then reduced by the peroxidase activity to PGH2. Based on the crystal structure of PGHS-1 arachidonate complex, it is now possible to envision how arachidonate is bound and oxygenation occurs. Recently, it has become possible to distinguish kinetically between the cyclooxygenase and peroxidase suicide inactivation reactions. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Cyclooxygenase; Peroxidase; Arachidonic acid; Eicosapentaenoic acid; Linoleic acid; 2-Arachidonylglycerol; Aspirin; Non-steroidal anti-inflammatory drugs; Celebrex; Rofecoxib; Ibuprofen; Aspirin
1. Introduction The biosynthesis of prostanoids, which include the prostaglandins (PGs) and thromboxanes, occurs in three steps: (a) the mobilization of a fatty acid substrate, typically arachidonic acid (AA), from membrane phospholipids through the action of a phospholipase A2 ; (b) Abbreviations: AA, arachidonic acid; PG, prostaglandin; PGHS, prostaglandin endoperoxide H synthase; EtOOH, ethyl hydrogen peroxide; TMPD, N,N,N ,N -tetramethylphenylenediamine; COX, cyclooxygenase; POX, peroxidase; NSAID, non-steroidal anti-inflammatory drug ∗ Corresponding author. Tel.: +1-517-355-1604; fax: +1-517-353-9334. E-mail address: [email protected] (W.L. Smith). 0090-6980/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 0 - 6 9 8 0 ( 0 2 ) 0 0 0 2 5 - 4
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Fig. 1. COX and POX reactions catalyzed by PGHSs.
the formation of PG endoperoxide H2 (PGH2 ) from AA mediated by a PG endoperoxide H synthase (PGHS) (Fig. 1); and (c) the conversion of PGH2 to a specific prostanoid through the action of a synthase such as PGE synthase. In this chapter, we summarize the enzymological properties of PGHSs, the enzymes that catalyze the second step, the committed step of the pathway. Several other recent and more detailed reviews are available on various aspects of this topic [1–6].
2. Interplay between cyclooxygenase and peroxidase activities of PGHSs There are two PGHS isozymes, PGHS-1 and -2 that have similar enzymological and structural properties. PGHSs exhibit two different but complementary enzymatic activities
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including a cyclooxygenase (COX) (bis-oxygenase) that catalyzes the conversion of AA and two molecules of O2 to PGG2 and a peroxidase (POX) that catalyzes the reduction of the 15-hydroperoxyl group of PGG2 (or 2-PGG2 -glycerol) to PGH2 (or 2-PGH2 -glycerol) (Fig. 1). The COX activity of PGHS-2, but not PGHS-1 can also use 2-arachidonyl glycerol (2-AG) as a substrate and convert it to 2-PGG2 -glycerol. The COX and POX activities occur at physically separate but functionally interactive locations within the protein. The model shown in Fig. 2A depicts the catalytic relationship between the COX and POX activities; the model in Fig. 2B depicts the spatial interrelationships between catalytically important residues. The mechanistic model in Fig. 2A was developed by Ruf and coworkers [7], and the basics of this model remains same today. In brief, the heme group at the POX site of PGHS undergoes a two electron oxidation by a hydroperoxide (e.g. PGG2 ) yielding the corresponding alcohol (e.g. PGH2 ) and an oxyferryl heme radical cation (compound I). In the next step, a tyrosinate residue (Tyr 385) contributes an election to compound I producing an oxyferryl heme and a tyrosyl radical (intermediate II). Finally, the tyrosyl radical abstracts a hydrogen from AA to begin the COX cycle of oxygen insertion and cyclization reactions. Neither the identity nor the source of the hydroperoxide necessary to initiate the first heme oxidation in vivo is known. In vitro, there is typically sufficient hydroperoxide in commercial fatty acid substrate preparations to initiate the process, and, once started, a hydroperoxide (e.g. PGG2 ) becomes available to continue the process as necessary. The POX reaction requires a reducing cosubstrate to convert compound I to II, and compound II to the heme of the resting enzyme. The identity of the reducing cosubstrate, in vivo, is not known. Interestingly, once the COX catalytic cycle has been initiated, it can operate independently of the POX cycle [8]. That is, the POX and COX reactions are not tightly coupled in the sense that there is a one to one correspondence between peroxide reduction and PGG2 formation [9]. Viewed from another perspective, the oxyferryl heme group of intermediate II, which is the same as the oxyferryl heme group of compound II of the POX cycle, can be reduced by one electron coming from a reducing cosubstrate to yield resting heme (Fe3+ -protoporphyrin IX) while the COX cycle continues to function [8].
3. PGHS peroxidase catalysis Although COX catalysis requires an initial oxidation of the heme group at the POX active site, POX catalysis can operate at or near maximal efficiency in the absence of COX turnover or occupancy of the COX active site [8,10,11]. PGHS POX has many of the same catalytic and spectroscopic properties of well-studied POXs including cytochrome c and horseradish POXs [12]. As noted by Garavito and coworkers [13] in the pioneering work on PGHS crystallography, the structure of PGHS-1 is closely related to that of myeloperoxidase; and, not unexpectedly, the structure of PGHS-2 is very similar to that of PGHS-1 [14,15]. POX rates are conveniently measured spectroscopically by monitoring changes in the absorbance of electron donors as they reduce oxidized heme intermediates formed when a hydroperoxide reacts with the heme group of the enzyme (Fig. 2A). N,N,N ,N -tetramethylphenylenediamine (TMPD) [16] and guaiacol [17] are the electron donors that have been used most widely in studying PGHS POX activities. These latter spectroscopic protocols
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Fig. 2. Interrelationships between COX and POX. (A) Mechanistic interrelationships between the COX and POX catalytic cycles. (B) Spatial relationships among catalytically important residues in COX and POX catalysis. AA, arachidonic acid; PPIX, protoporphorin IX; ROOH, an alkyl hydroperoxide (e.g. PGG2 ); ROH, an alkyl alcohol; I, compound I; II, compound II.
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measure the overall rate of the POX reaction. However, it is also possible to measure the rates of formation of the individual intermediates in POX catalysis (i.e. compounds I and II) using stopped flow spectrophotometry on a millisecond time scale. A characteristic of POXs is that they form spectroscopically identifiable oxidized heme intermediates during their catalytic cycles. As discussed before, the heme group at the POX active site of PGHS reacts with a hydroperoxide to form compound I [18–20]. Native PGHS-1 has a Soret peak for resting heme at 410 nm which is reduced in intensity and red shifted in forming compound I with an isosbestic point at 414 nm. This reaction, although rate limiting in POX catalysis, is actually relatively rapid with second order rate constants for hydroperoxide substrates in the range of 107 mol−1 s−1 ; in practical terms this means that when PGHS-1 is incubated with a small excess of a hydroperoxide substrate such as ethylhydrogen peroxide at concentrations in the millimolar range, compound I formation is complete within 10 ms at 4 ◦ C. In the presence of exogenous electron donors such as guaiacol or TMPD, compound I undergoes two very rapid, successive one-electron reductions, resulting in the formation of compound II and then resting enzyme (Fig. 2). PGHS-1 compound II has a prominent Soret peak at 424 nm [20]. Although Fe3+ -protoporphyrin IX is the natural heme ligand, Mn3+ -heme, but not other heme forms [21], will substitute in POX catalysis by PGHSs forming higher oxidation states of Mn3+ analogous to those seen with Fe3+ [22–24]. The specific activity of the Mn3+ -heme form of PGHS-1 is about 5% that of the Fe3+ -heme form.
4. Peroxidase substrate specificities The POX activities of PGHSs can reduce a variety of peroxides, but both isozymes, PGHS-1 and -2, show a preference for secondary alkyl hydroperoxides, among which is PGG2 , probably the physiologically most important substrate [20,25]. PGHS-1 POX activity has relatively low activity with tertiary hydroperoxides such as cumene hydroperoxide and t-butyl-hydroperoxide [26]. In vitro, relatively hydrophobic alkyl hydroperoxides such as 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP) exhibit about 10-fold higher secondary rate constants for formation of compound I than ethylhydroperoxide [20] about 1000-fold higher rate constants than observed with H2 O2 . Lower apparent Km values for the POX reaction as measured by rates of oxidation of reducing cosubstrates are also observed for relatively hydrophobic primary and secondary hydroperoxides (e.g. ∼10 versus 300 M for H2 O2 ) [20,25]. The PGHS POX reaction occurs at a heme-containing active site that appears in crystal structures to be near the protein surface and relatively exposed to solvent [13–15]. Therefore, the hydroperoxide substrate specificity is difficult to rationalize based on crystal structures. Moreover, while cyanide binds reasonably tightly to the heme group and in a linear orientation [27], the slightly larger azide group does not tightly (Seibold, unpublished data); this low affinity for azide is surprising because, again, there is no “ceiling” over the POX active site in the crystal structures of either PGHS-1 of PGHS-2. PGHS POX activity has relatively little specificity toward reducing cosubstrates [12,17,28]. However, two points of interest are that ascorbate is a poor reductant and that hydroperoxides themselves can function as reducing cosubstrates, which can cause problems in interpreting reaction kinetics [29].
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Residues thought to be involved in coordination of the heme iron or in POX catalysis have been examined by mutagenic analyses [25,27,30]. The heme irons of most POXs are coordinated by the four nitrogens of the protoporphyrin ring and at the fifth coordination position by the N␦ atoms of the imidazole group of the proximal histidine. In some cases, the iron is also coordinated at the sixth position either with a small inorganic ion or water. The classic push–pull hypothesis used to rationalize POX catalysis by heme POXs is that the distal histidine located near the sixth coordination position pulls a proton from the peroxide substrate as one of the oxygen atoms binds the sixth coordination position of the heme iron while a strongly basic, relatively electron-rich imidazolate anion of the proximal histidine ligand helps stabilize higher oxidation states of iron during oxidation of the heme [31]. Based on this hypothesis, the distal histidine of PGHS-1, His 207, would be predicted to be important in the deprotonation of the hydroperoxide substrate and subsequent protonation of the incipient alkoxide ion to form the alcohol during generation of compound I [25,30] and His 388, the proximal heme ligand, would be expected to have considerable anionic character. Additionally, Gln 203 would be considered to be important in facilitating heterolytic cleavage of the oxygen–oxygen bond by stabilizing the negative charge developing on the incipient alkoxide ion. Not surprisingly, results from site directed mutatgenesis indicate that indeed His 207, Gln 203 and His 388 are important in catalysis [25,27,30]. The results of studies of mutants of His 207 are consistent with its role as the distal histidine. However, the results of studies of mutants of Gln 203 and His 388 are not entirely consistent with their predicted roles in catalysis. Substitution of Gln 203 with arginine eliminates POX activity despite the fact that one might expect an arginine at this position to stabilize the alkoxide [25]; moreover, conversion of Gln 203 to an asparagine yields a mutant enzyme that is quite active although having a smaller residue at position 203 would be expected for steric reasons to be considerable less effective in charge stabilization. Not unexpectedly, H388Q and H388A mutants of PGHS-1 are inactive [30]. As noted before His 388 is the proximal (fifth) heme ligand with its N␦ imidazole nitrogen bonded to Fe3+ ; the Nε imidazole nitrogen may bond a water molecule that, in turn, may bond Tyr 504 [27]. In most heme POXs including myeloperoxidase, the proximal histidine interacts with an aspartate group causing the imidazole to be relatively basic (i.e. anionic), which increases the strength of the bond between the heme Fe3+ and the N␦ of the imidazole drawing the iron out of the heme plane and favoring a five-coordinate state. However, crystallographic data and visible and resonance Raman spectroscopy of resting oPGHS-1 indicate that it is mainly in the high-spin ferric form with Fe3+ in the plane of the ring and a water occupying the distal (sixth) coordination position [27]. Furthermore, resonance Raman spectroscopy of the complex between CO and the reduced enzyme (i.e. Fe2+ heme) indicate that imidazole group of His 388 is neutral, not anionic. Finally, substitution of Tyr 504 with alanine, a change that would disrupt any H2 O-mediated interaction between Tyr 504 and His 388, had little effect on PGHS-1 POX activity [27]. The generally accepted push–pull mechanism for POX catalysis requires strong basicity of the proximal ligand [32], His 388 in case of PGHSs. Collectively however, these recent studies on the heme–His 388 interaction in PGHS-1 imply that a strongly basic proximal ligand is not necessary for PGHS POX catalysis and that the “push” feature of the proposed mechanism for POX catalysis does not apply to PGHS-1 [27]. A neutral proximal histidine has also been observed two marine worm POXs [33].
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5. Comparisons of the peroxidase activities of PGHS-1 and -2 There are two significant differences between the POX activities of PGHS-1 and -2. First, PGHS-1 catalyzes primarily a standard two-electron reduction of hydroperoxide involving heterolytic cleavage of the peroxide group and yielding compound I and an alcohol; in contrast, PGHS-2 catalyzes 60% two-electron and 40% one-electron reductions [25]. The abilities of native and mutant PGHS-2 to catalyze heterolytic cleavage of peroxides correlate with their abilities to catalyze the COX reaction, which is consistent with compound I being the precursor of intermediate II (Fig. 2A) [25]. It is unclear if there is any biological significance to homolytic cleavage of hydroperoxides catalyzed by PGHS-2, and there is no obvious structural reason why PGHS-2 tends to produce close to 50% homolytic cleavage. A second significant difference between the POX activities of PGHS-1 and -2 is that the rate of formation of intermediate II formation from compound I is much faster with PGHS-2 [20]. This accounts in part for the fact that for PGHS-2, intermediate II is formed more rapidly and at lower peroxide concentrations. As a consequence of this more facile formation of intermediate II with its associated tyrosyl radical, PGHS-2 COX activity is activated at lower concentrations of hydroperoxide than those required to activate PGHS-1 [34,35]. This is potentially important biologically in that under conditions of low “peroxide tone” in cells, PGHS-2 could function in the presence of PGHS-1 without PGHS-1 being able to function [2,6,20]. Once again, there is no obvious structural explanation for the differences in rates of intermediate II formation property by PGHS-1 versus PGHS-2. 6. PGHS peroxidase inactivation PGHS POX activity is lost during reaction with various hydroperoxides. This inactivation process is a mechanism-based, suicide inactivation with t1/2 values in the range of 2–20 s depending on the reaction conditions [36,37]. The process occurs independent of the structure of the peroxide or occupancy of the COX active site [11,37,38]. Stopped flow spectroscopic and kinetic data originally suggested that POX inactivation involves a sequential conversion of compound I to COX intermediate II to III to a dead-end species with the slow step in the process being the last step-formation of the inactive enzyme [37,38]. More recently, however, it has become clear that the intermediate II in this process is actually an intermediate IIA with a tyrosyl radical being localized to a residue other than Tyr 385 (Fig. 2A) [11,39–41]. The nature of the protein modification(s) associated with suicide inactivation has not been defined. 7. Future directions in peroxidase research The POX activity of PGHS has not been studied in nearly so much detail as the COX activity of the enzyme so numerous questions remain. Two key areas of investigation focus on identifying the intermediates involved in the formation of both compound I and intermediate II and the basis for the unusual oxidant substrate specificity of PGHS POX. Studies in various laboratories are using molecular modeling, crystallographic, spectroscopic, and mutagenic approaches to address these questions.
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8. Cyclooxygenase catalysis The initial and rate limiting step in the COX reaction is the stereospecific removal of the 13-proS hydrogen from AA which yields an AA radical [42–44]. As depicted in Fig. 2B, an AA molecule becomes oriented in the COX active site with C-13 neighboring Tyr 385. Subsequent crystallographic analyses of AA in the active site of PGHS-1, that are described in further detail elsewhere in this compendium, indicate that the distance between the phenolic oxygen of Tyr 385 and the 13-proS hydrogen is about 2.5 Å [45]. This is very important because it indicates that AA is in a catalytically competent orientation (i.e. one that is converted to an oxygenated product) in the AA/PGHS-1 crystal. The AA radical formed in the rate limiting step has an unpaired electron centered initially on C-13. This radical isomerizes and the O2 diradical reacts with the AA radical having the electron centered at C-11 to yield the 11R-hydroperoxyl radical. In solution, a double allylic, carbon-centered radical formed at C-13 would rearrange to provide three major sites for O2 insertion at C-11, -13, and -15, yet greater than 97% of the O2 addition occurs at C-11 [46,47]. It is uncertain why this is so. It may be principally steric. There is more space for an O2 molecule under C-11 than near C-13 or -15 in the AA/PGHS-1 complex [45]. Alternatively or in addition, the strain existing in the conformation of the AA at the moment of hydrogen abstraction may cause the highest spin density to occur at C-11.1 Hydrogen abstraction and O2 insertion at C-11 do not require any change in the structure of AA [45]. However, formation of the carbon–carbon bond between C-8 and -12 requires that these atoms come within 1.5 Å, and in the AA crystal structure the distance between C-8 and -12 is about 4.5 Å. We have proposed that this occurs by rotation about the C-10/C-11 bond with the C-11 hydroperoxyl radical then adding to the back (top) side of C-9 to form the endoperoxide concomitant with movement of C-12 toward C-8 to form the cyclopentane ring [45]. Support for this concept comes from a crystal structure of the endoperoxide analog U44619 in the PGHS-1 COX site that shows from C-1 to -7 of this analog located in approximately the same positions as C-1 to -7 of AA and the hydroxyl group at C-15 of the analog located adjacent to Tyr 385.2 In fact, this latter observation suggests (a) that C-15 does move nearer the Tyr 385 position presumably favoring the second O2 addition at C-15, and (b) that in the PGG2 radical having the 15-hydroperoxyl radical substituent, that the 15-hydroperoxyl group can reabstract the hydrogen from Tyr 385 thereby regenerating the tyrosyl radical and yielding the PGG2 product. The 15-hydroperoxyl group of PGG2 is subsequently reduced to PGH2 by the POX activity of PGHSs. Based on the crystal structure, there is no direct route for PGG2 to travel through the protein from the COX to the POX site. Presumably, PGG2 exits the COX site through the opening in the membrane binding domain and travels around the surface of the protein to its site of reduction. 9. Cyclooxygenase substrate specificity PGHS-1 appears to use fatty acids such as arachidonate exclusively as substrates whereas PGHS-2 utilizes both fatty acids and 2-AG about equally well [48,49]. The ability of PGHS-2 1 2
Porter, personal communication. Malkowski, unpublished observation.
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to utilize alternative substrates may explain why there are two isozymes [6,48,49]; that is, one enzyme can generate a group of products that cannot be generated via the other isoform [50]. Arachidonate is the preferred fatty acid substrate for both PGHS-1 and -2. However, both enzymes will also oxygenate various n−3 and n−6 C18 and C20 fatty acids in vitro with catalytic efficiencies in the range of 0.05–0.7 of that of arachidonate [51]. At least some of these substrates including 9,12-octadecadienoate (18:2n−6), 8,11,14-eicosatrienoate (20:3n−6) and 5,8,11,14,17-eicosapentaenoate (20:5n−3) are oxygenated via COX activity when added exogenously to intact cells [52,53] or when mobilized from cellular phosphoglycerides [54–58]. In vesicular gland which has low levels of arachidonate, homo-␥-linolenic acid (20:3n−6) is converted efficiently to 1-series prostanoid products found in abundance in semen [56]. Other fatty acids that are COX substrates include adrenic acid (22:4n−6) [59], the Mead acid 5Z,8Z,11Z-eicosatrienoic acid [60], columbinic acid (5E,9Z,12Z-octadecatrienoic acid) [61,62], and 5,6-oxido-eicosatrienoic acid [63,64]. Substrates other than arachidonate typically have somewhat lower Km values than arachidonate but can compete with arachidonate for the COX active site thereby inhibiting formation of 2-series prostanoids [65]. This inhibition may be important in vivo in regulating overall prostanoid formation [66–68].
10. Multiple catalytically competent forms of substrates Closely related to the overall issue of substrate specificity is a subtle issue of the existence of different catalytically competent forms of the same substrate. PGHS-1 and -2 are both capable of forming more than one product from AA [46,69] and other substrate fatty acids [42]. PGHS-1 produces PGG2 , 11R-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid (11R-HETE), 15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15S-HETE) and 15R-HETE from AA. At saturating substrate concentrations about 95% of the product is PGG2 . Thus, there are obvious differences in the Vmax values for the formation of these products. Additionally, there are differences in the Km values (e.g. 5.5, 12, and 19 M for PGG2 , 11-HETE and 15-HETE formation, respectively) [46]. However, the KI values for inhibition of the formation of these different products by a competitive inhibitor are the same. These data are most easily interpretable as arachidonate being able to assume at least three catalytically productive arrangements within the COX site of PGHS-1.
11. Cyclooxygenase kinetics and their measurement There are three general methods for quantifying COX activity: (a) an O2 electrode assay for measuring O2 consumption involved in the conversion of arachidonate to PGG2 , (b) a radio-thin layer chromatography assay for measuring the conversion of 14 C-arachidonate to prostanoids, and (c) a sensitive luminescence assay which depends on the coordinate actions of COX and POX activities [70]. Note that for accurate measurements of O2 consumption corrections must be made for damping of electrode responses [9,11] and for suicide inactivation and peroxide activation [36,71].
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12. Cyclooxygenase inhibition by non-steroidal anti-inflammatory drugs PGHS-1 and -2 are the major pharmacological targets of non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs are competitive COX inhibitors which interfere with fatty acid substrate binding. Classical NSAIDs such as ibuprofen and naproxen inhibit the COX activity of both PGHS-1 and -2. The newly developed rofecoxib and celecoxib are more selective for PGHS-2. These latter drugs were developed to be anti-inflammatory and analgesic without causing the gastrointestinal side effects of classical NSAIDs. The structural basis for NSAID actions have been reviewed in detail elsewhere [3,5]. The most notable points at the enzymological level are the structural differences between the COX sites of the two isozymes that have permitted the development of isoform selective inhibitors [14,15] and the somewhat unusual pharmacological profiles that have been observed for the interactions the various drugs with the COXs [3,5,72]. Most notably, the new COX-2 inhibitors exhibit PGHS-2 selectivity because they inhibit this isoform by a time-dependent, pseudo-irreversible mechanism, while they inhibit PGHS-1 by a rapid, competitive, and reversible mechanism [3,5,72]. A detailed treatment of the topic of NSAIDs and a discussion of the mechanism of aspirin inhibition can be found elsewhere [3,5,72].
13. Cyclooxygenase inactivation Both the COX and POX activities of PGHS are inactivated during catalysis as the result of a non-productive breakdown of active enzyme intermediates. One COX active site is lost per about 2000 catalytic turnovers. The chemical changes in the protein that accompany these two suicide inactivation processes are unknown. The changes presumably involve reaction of amino acid radicals (e.g. tyrosyl radicals) with other groups on the protein and then reaction with molecular oxygen. Suicide inactivation is a crude regulatory mechanism which places an upper limit on cellular PG biosynthetic activity. It is unlikely that the intermediate(s) responsible for COX inactivation are substrate-derived radicals because the rate of covalent attachment of fatty acid to PGHS during catalysis is 30 times slower than that of suicide inactivation [73,74]. Interestingly, a Y385F oPGHS-1 mutant undergoes POX suicide inactivation at approximately the same rate as native PGHS-1 [11]; additionally, more than half of the POX activity can be retained following complete loss of COX activity under certain conditions [11]. COX inactivation involves COX intermediate II whereas POX inactivation occurs via intermediate IIA (Fig. 2A).
14. Future directions in cyclooxygenase research Three areas that are likely to be targets for future studies of COX activity are (a) chemical characterization of the process of COX suicide inactivation, (b) determination of the mechanism of formation of intermediate II from compound I, and (c) identification of the catalytically competent forms of AA that yield the 11R-HETE and 15R/S-HETE, respectively.
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Acknowledgements Work in the authors’ laboratories that was mentioned in this review was supported by NIH Grants DK22042 and GM57323. References [1] Marnett LJ, Rowlinson SW, Goodwin DC, Kalgutkar AS, Lanzo CA. Arachidonic acid oxygenation by COX-1 and COX-2. J Biol Chem 1999;274:22903–6. [2] Kulmacz RJ. Cellular regulation of prostaglandin H synthase catalysis. FEBS Lett 1998;430:154–7. [3] Smith WL, DeWitt DL, Garavito RM. Cyclooxygenases: structural, cellular and molecular biology. Annu Rev Biochem 2000;69:149–82. [4] DeWitt DL. Cox-2-selective inhibitors: the new super aspirins. Mol Pharmacol 1999;55:625–31. [5] Marnett LJ, Kalgutkar AS. Cyclooxygenase 2 inhibitors: discovery, selectivity and the future. Trends Pharmacol Sci 1999;20:465–9. [6] Smith WL, Langenbach R. Why there are two cyclooxygenases? J Clin Invest 2001;107:1491–5. [7] Dietz R, Nastainczyk W, Ruf HH. Higher oxidation states of prostaglandin H synthase. Rapid electronic spectroscopy detected two spectral intermediates during the peroxidase reaction with prostaglandin G2. Eur J Biochem 1988;171:321–8. [8] Koshkin V, Dunford HB. Coupling of the peroxidase and cyclooxygenase reactions of prostaglandin H synthase. Biochem Biophys Acta 1999;1430:341–8. [9] Wei C, Kulmacz RJ, Tsai AL. Comparison of branched-chain and tightly coupled reaction mechanisms for prostaglandin H synthase. Biochemistry 1995;34:8499–512. [10] Mizuno K, Yamamoto S, Lands WEM. Effects of non-steroidal anti-inflammatory drugs on fatty acid cyclooxygenase and prostaglandin hydroperoxidase activities. Prostaglandins 1982;23:743–57. [11] Song I, Ball TM, Smith WL. Different suicide inactivation processes for the peroxidase and cyclooxygenase activities of prostaglandin endoperoxide H synthase-1. Biochem Biophys Res Commun, in press. [12] Smith WL, Marnett LJ. Prostaglandin endoperoxide synthases. In: Sigel H, Sigel A, editors. Metal ions in biological systems, vol. 30. New York: Marcel Dekker, 1994. p. 163–99. [13] Picot D, Loll PJ, Garavito M. The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature 1994;367:243–9. [14] Luong C, Miler A, Barnett J, Chow J, Ramesha C, Browner MF. Flexibility of the NSAID binding site in the structure of human cylcooxygenase-2. Nat Struct Biol 1996;3:927–33. [15] Kurumbail RG, Stevens AM, Gierse JK, McDonald JJ, Stegeman RA, Pak JY, et al. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nature 1996;384:644–8. [16] Kulmacz RJ. Prostaglandin G2 levels during reaction of prostaglandin H synthase with arachidonic acid. Prostaglandins 1987;34:225–40. [17] Markey CM, Alward A, Weller PE, Marnett LJ. Quantitative studies of hydroperoxide reduction by prostaglandin H synthase. Reducing substrate specificity and the relationship of peroxidase to cyclooxygenase activities. J Biol Chem 1987;262:6266–79. [18] Lambeir AM, Markey CM, Dunford HB, Marnett LJ. Spectral properties of the higher oxidation states of prostaglandin H synthase. J Biol Chem 1985;260:14894–6. [19] Karthein R, Dietz R, Nastainczyk W, Ruf HH. Higher oxidation states of prostaglandin H synthase. EPR study of a transient tyrosyl radical in the enzyme during the peroxidase reaction. Eur J Biochem 1988;171:313–20. [20] Lu G, Tsai A-L, Van Wart HE, Kulmacz RJ. Comparison of the peroxidase reaction kinetics of prostaglandin H synthase-1 and -2. J Biol Chem 1999;274:16162–7. [21] Ogino N, Ohki S, Yamamoto S, Hayaishi O. Prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. Inactivation and activation by heme and other metalloporphyrins. J Biol Chem 1978;253:5061–8. [22] Tsai A, Wei C, Baek HK, Kulmacz RJ, Van Wart HE. Comparison of peroxidase reaction mechanisms of prostaglandin H synthase-1 containing heme and mangano protoporphyrin IX. J Biol Chem 1997;272: 8885–94.
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[23] Odenwaller R, Maddipati KR, Marnett LJ. Detection of a higher oxidation state of manganese-prostaglandin endoperoxide synthase. J Biol Chem 1992;267:13863–9. [24] Strieder S, Schaible K, Scherer HJ, Dietz R, Ruf HH. Prostaglandin endoperoxide synthase substituted with manganese protoporphyrin IX. Formation of a higher oxidation state and its relation to cyclooxygenase reaction. J Biol Chem 1992;267:13870–8. [25] Landino LM, Crews BC, Gierse JK, Hauser SC, Marnett LJ. Mutational analysis of the role of the distal histidine and glutamine residues of prostaglandin-endoperoxide synthase-2 in peroxidase catalysis, hydroperoxide reduction, and cyclooxygenase activation. J Biol Chem 1997;272:21565–74. [26] Ohki S, Ogino N, Yamamoto S, Hayaishi O. Prostaglandin hydroperoxidase, an integral part of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J Biol Chem 1979;254:829–36. [27] Seibold SA, Cerda JF, Mulichak AM, Song I, Garavito RM, Arakawa T, Smith WL, Babcock GT. Peroxidase activity in prostaglandin endoperoxide H synthase-1 occurs with a neutral histidine proximal heme ligand. Biochemistry 2000;39:6616–24. [28] Hsuanyu Y, Dunford HB. Prostaglandin H synthase kinetics: the effect of substituted phenols on cyclooxygenase activity and the substituent effect on phenolic peroxidatic activity. J Biol Chem 1992;267:17649–57. [29] Hsuanyu Y, Dunford HB. Kinetics of the reaction of prostaglandin H synthase compound II with ascorbic acid. Arch Biochem Biophys 1990;281:282–6. [30] Shimokawa T, Smith WL. Essential histidines of prostaglandin endoperoxide synthase. J Biol Chem 1991;266:6168–73. [31] Poulos TL, Kraut J. The stereochemistry of peroxidase catalysis. J Biol Chem 1980;255:8199–205. [32] Dawson JH. Probing structure-function relations in heme-containing oxygenases and peroxidases. Science 1988;240:433–9. [33] Franzen S, Roach MP, Chen YP, Dyer B, Woodruff WH, Dawson JH. The unusual reactivities of Amphitrite ornata dehaloperoxidase and Notomastus lobatus chloroperoxidase do not arise from a histidine imidazolate proximal heme iron ligand. J Am Chem Soc 1998;8120:4658–61. [34] Capdevila JH, Morrow JD, Belosludtsev YY, Beauchamp DR, DuBois RN, Falck JR. The catalytic outcomes of the constitutive and the mitogen inducible isoforms of prostaglandin H2 synthase are markedly affected by glutathione and glutathione peroxidase(s). Biochemistry 1995;34:3325–37. [35] Kulmacz RJ, Wang LH. Comparison of hydroperoxide initiator requirements for the cyclooxygenase activities of prostaglandin H synthase-1 and -2. J Biol Chem 1995;270:24019–23. [36] Callan OH, So OY, Swinney DC. The kinetic factors that determine the affinity and selectivity for slow binding inhibition of human prostaglandin H synthase 1 and 2 by indomethacin and flurbiprofen. J Biol Chem 1996;271:3548–54. [37] Wu G, Wei C, Kulmacz RJ, Osawa Y, Tsai A-L. A mechanistic study of self-inactivation of the peroxidase activity in prostaglandin H synthase-1. J Biol Chem 1999;274:9231–7. [38] Wu G, Vuletich JL, Kulmacz RJ, Osawa Y, Tsai A-L. Peroxidase self-inactivation in prostaglandin H synthase-1 pretreated with cyclooxygenase inhibitors or substituted with mangano protoporphyrin IX. J Biol Chem 2001;276:19879–88. [39] Tsai A, Hsi LC, Kulmacz RJ, Palmer G, Smith WL. Characterization of the tyrosyl radicals in ovine prostaglandin H synthase-1 by isotope replacement and site-directed mutagenesis. J Biol Chem 1994;269:5085–91. [40] Hsi LC, Hoganson CW, Babcock GT, Garavito RM, Smith WL. An examination of the source of the tyrosyl radical in ovine prostaglandin endoperoxide synthase-1. Biochem Biophys Res Commun 1995;207:652–60. [41] Shi W, Hoganson CW, Espe M, Bender CJ, Babcock GT, Palmer G, et al. Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1. Biochemistry 2000;39:4112–21. [42] Hamberg M, Samuelsson B. On the mechanism of the biosynthesis of prostaglandins E-1 and F-1-alpha. J Biol Chem 1967;242:5336–43. [43] Tsai A, Kulmacz RJ, Palmer G. Spectroscopic evidence for reaction of prostaglandin H synthase-1 tyrosyl radical with arachidonic acid. J Biol Chem 1995;270:10503–8. [44] Tsai A, Palmer G, Xiao G, Swinney DC, Kulmacz RJ. Structural characterization of arachidonyl radicals formed by prostaglandin H synthase-2 and prostaglandin H synthase-1 reconstituted with mangano protoporphyrin IX. J Biol Chem 1998;273:3888–94.
W.L. Smith, I. Song / Prostaglandins & other Lipid Mediators 68–69 (2002) 115–128
127
[45] Malkowski MG, Ginell S, Smith WL, Garavito RM. The X-ray crystal structure of prostaglandin endoperoxide H synthase-1 complexed with arachidonic acid. Science 2000;289:1933–7. [46] Thuresson ED, Lakkides KM, Smith WL. Different catalytically competent arrangements of arachidonic acid within the cyclooxygenase active site of prostaglandin endoperoxide H synthase-1 lead to the formation of different oxygenated products. J Biol Chem 2000;275:8501–7. [47] Thuresson ED, Lakkides KM, Rieke CJ, Sun Y, Wingerd BA, Micielli R, et al. Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid. J Biol Chem 2001;276:10347–57. [48] Kozak KR, Rowlinson SW, Marnett LJ. Oxygenation of the endocannabinoid, 2-arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2. J Biol Chem 2000;275:33744–9. [49] Kozak KR, Prusakiewicz JJ, Rowlinson SW, Schneider C, Marnett LJ. Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid 2-arachidonylglycerol. J Biol Chem 2001;276: 30072–7. [50] Kozak KR, Crews BC, Ray JL, Tai H-H, Morrow JD, Marnett LJ. Metabolism of prostaglandin glycerol esters and prostaglandin ethanolamides in vitro and in vivo. J Biol Chem 2001;276:30072–7. [51] Laneuville OI, Breuer DK, Xu N, Huang ZH, Gage DA, Watson JT, et al. Fatty acid substrate specificities of human prostaglandin H synthases-1 and -2: formation of 12 hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic acids from alpha-linolenic acid. J Biol Chem 1995;270:19330–6. [52] Baer AN, Costello PB, Green FA. Stereospecificity of the hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids produced by cultured bovine endothelial cells. Biochim Biophys Acta 1991;1085: 45–52. [53] Kaduce TL, Figard PH, Leifur R, Spector AA. Formation of 9-hydroxyoctadecadienoic acid from linoleic acid in endothelial cells. J Biol Chem 1989;264:6823–30. [54] Baer AN, Costello PB, Green FA. Stereospecificity of the products of the fatty acid oxygenases derived from psoriatic scales. J Lipid Res 1991;32:341–7. [55] Abeywardena MY, Fischer S, Schweer H, Charnock JS. In vivo formation of metabolites of prostaglandins I2 and I3 in the marmoset monkey (Callithrix jacchus) following dietary supplementation with tuna fish oil. Biochim Biophys Acta 1989;1003:161–6. [56] Knapp HR. Prostaglandins in human semen during fish oil ingestion: evidence for in vivo cyclooxygenase inhibition and appearance of novel trienoic compounds. Prostaglandins 1990;39:407–23. [57] Leaver HA, Howie A, Wilson NH. The biosynthesis of the 3-series prostaglandins in rat uterus after alpha-linolenic acid feeding: mass spectroscopy of prostaglandins E and F produced by rat uteri in tissue culture. Prostaglandins Leukot Essent Fatty Acids 1991;42:217–24. [58] Engels F, Willems H, Nijkamp FP. Cyclooxygenase-catalyzed formation of 9-hydroxylinoleic acid by guinea pig alveolar macrophages under non-stimulated conditions. FEBS Lett 1986;209:249–53. [59] Larsen LN, Dahl E, Bremer J. Peroxidative oxidation of leuco-dichlorofluorescein by prostaglandin H synthase in prostaglandin biosynthesis from polyunsaturated fatty acids. Biochim Biophys Acta 1996;1299:47–53. [60] Oliw EH, Hornsten L, Sprecher H, Hamberg M. Oxygenation of 5,8,11-eicosatrienoic acid by prostaglandin endoperoxide synthase and by cytochrome P450 monooxygenase: structure and mechanism of formation of major metabolites. Arch Biochem Biophys 1993;305:288–97. [61] Elliott WJ, Morrison AR, Sprecher HW, Needleman P. The metabolic transformations of columbinic acid and the effect of topical application of the major metabolites on rat skin. J Biol Chem 1985;260:987–92. [62] Nugteren DH, Christ Hazelhof E. Naturally occurring conjugated octadecatrienoic acids are strong inhibitors of prostaglandin biosynthesis. Prostaglandins 1987;33:403–17. [63] Oliw EH. Metabolism of 5(6)oxidoeicosatrienoic acid by ram seminal vesicles. Formation of two stereoisomers of 5-hydroxyprostaglandin I1. J Biol Chem 1984;259:2716–21. [64] Balazy M. Metabolism of 5,6-epoxyeicosatrienoic acid by the human platelet. Formation of novel thromboxane analogs. J Biol Chem 1991;266:23561–7. [65] Lands WEM, LeTellier PR, Rome LH, Vanderhoek JY. Inhibition of prostaglandin biosynthesis. Adv Biosci 1973;9:15–28. [66] Needleman P, Whitaker MO, Wyche A, Watters K, Sprecher H, Raz A. Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: pharmacological basis for a therapeutic approach. Prostaglandins 1980;19:165–81.
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[67] Spector AA, Kaduce TL, Figard PH, Norton KC, Hoak JC, Czervionke RL. Eicosapentaenoic acid and prostacyclin production by cultured human endothelial cells. J Lipid Res 1983;24:1595–604. [68] Lagarde M, Drouot B, Guichardant M, Dechavanne M. Uptake and effect on arachidonic acid oxygenation of some icosaenoic acids in human platelets. Biomed Biochim Acta 1984;43:S319–22. [69] Xiao G, Tsai AL, Palmer G, Boyar WC, Marshall PJ, Kulmacz RJ. Analysis of hydroperoxide-induced tyrosyl radicals and lipoxygenase activity in aspirin-treated human prostaglandin H synthase-2. Biochemistry 1997;36:1836–45. [70] Laneuville O, Manke CR, DeWitt DL, Smith WL. Assays for cyclooxygenase inhibiton by nonsteroidal anti-inflammatory drugs. In: Allison AC, editor. Weir’s handbook of experimental immunology, vol. IV. 5th ed. Oxford, UK: Blackwell 1995. p. 1–6. [71] Chen W, Pawelek TR, Kulmacz RJ. Hydroperoxide dependence and cooperative cyclooxygenase kinetics in prostaglandin H synthase-1 and -2. J Biol Chem 1999;274:20301–6. [72] Smith WL, DeWitt DL. Prostaglandin endoperoxide H synthases-1 and -2. In: Dixon FJ, editor. Advances in immunology, vol. 62. San Diego, CA: Academic Press 1996. p. 167–215. [73] Kulmacz RJ. Attachment of substrate metabolite to prostaglandin H synthase upon reaction with arachidonic acid. Biochem Biophys Res Commun 1987;148:539–45. [74] Lecomte M, Lecocq R, Dumont JE, Boeynaems J-M. Covalent binding of arachidonic acid metabolites to human platelet proteins. J Biol Chem 1990;265:5178–87.
The enzymology of prostaglandin endoperoxide H synthases-1 and -2 William L. Smith a,∗ , Inseok Song b a
Department of Biochemistry and Molecular Biology, Michigan State University, 513 Biochemistry Building, East Lansing, MI 48824 USA b Department of Life Science, University of Seoul, Seoul, South Korea
Abstract We summarize the enzymological properties of prostaglandin endoperoxide H synthases (PGHs)-1 and -2, the enzymes that catalyze the committed step in prostaglandin biosynthesis. These isoenzymes are closely related structurally and mechanistically. Each catalyzes a peroxidase and a cyclooxygenase reaction at spatially separate but neighboring, electronically interrelated active sites. The peroxidase is necessary to activate the cyclooxygenase; oxidation of the heme group of the peroxidase by peroxide leads to oxidation of a cyclooxygenase active site tyrosine. The tyrosine radical abstracts hydrogen from arachidonic acid to form an arachidonate radical which reacts sequentially with two oxygen molecules forming the intermediate product PGG2. PGG2 is then reduced by the peroxidase activity to PGH2. Based on the crystal structure of PGHS-1 arachidonate complex, it is now possible to envision how arachidonate is bound and oxygenation occurs. Recently, it has become possible to distinguish kinetically between the cyclooxygenase and peroxidase suicide inactivation reactions. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Cyclooxygenase; Peroxidase; Arachidonic acid; Eicosapentaenoic acid; Linoleic acid; 2-Arachidonylglycerol; Aspirin; Non-steroidal anti-inflammatory drugs; Celebrex; Rofecoxib; Ibuprofen; Aspirin
1. Introduction The biosynthesis of prostanoids, which include the prostaglandins (PGs) and thromboxanes, occurs in three steps: (a) the mobilization of a fatty acid substrate, typically arachidonic acid (AA), from membrane phospholipids through the action of a phospholipase A2 ; (b) Abbreviations: AA, arachidonic acid; PG, prostaglandin; PGHS, prostaglandin endoperoxide H synthase; EtOOH, ethyl hydrogen peroxide; TMPD, N,N,N ,N -tetramethylphenylenediamine; COX, cyclooxygenase; POX, peroxidase; NSAID, non-steroidal anti-inflammatory drug ∗ Corresponding author. Tel.: +1-517-355-1604; fax: +1-517-353-9334. E-mail address: [email protected] (W.L. Smith). 0090-6980/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 0 - 6 9 8 0 ( 0 2 ) 0 0 0 2 5 - 4
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Fig. 1. COX and POX reactions catalyzed by PGHSs.
the formation of PG endoperoxide H2 (PGH2 ) from AA mediated by a PG endoperoxide H synthase (PGHS) (Fig. 1); and (c) the conversion of PGH2 to a specific prostanoid through the action of a synthase such as PGE synthase. In this chapter, we summarize the enzymological properties of PGHSs, the enzymes that catalyze the second step, the committed step of the pathway. Several other recent and more detailed reviews are available on various aspects of this topic [1–6].
2. Interplay between cyclooxygenase and peroxidase activities of PGHSs There are two PGHS isozymes, PGHS-1 and -2 that have similar enzymological and structural properties. PGHSs exhibit two different but complementary enzymatic activities
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including a cyclooxygenase (COX) (bis-oxygenase) that catalyzes the conversion of AA and two molecules of O2 to PGG2 and a peroxidase (POX) that catalyzes the reduction of the 15-hydroperoxyl group of PGG2 (or 2-PGG2 -glycerol) to PGH2 (or 2-PGH2 -glycerol) (Fig. 1). The COX activity of PGHS-2, but not PGHS-1 can also use 2-arachidonyl glycerol (2-AG) as a substrate and convert it to 2-PGG2 -glycerol. The COX and POX activities occur at physically separate but functionally interactive locations within the protein. The model shown in Fig. 2A depicts the catalytic relationship between the COX and POX activities; the model in Fig. 2B depicts the spatial interrelationships between catalytically important residues. The mechanistic model in Fig. 2A was developed by Ruf and coworkers [7], and the basics of this model remains same today. In brief, the heme group at the POX site of PGHS undergoes a two electron oxidation by a hydroperoxide (e.g. PGG2 ) yielding the corresponding alcohol (e.g. PGH2 ) and an oxyferryl heme radical cation (compound I). In the next step, a tyrosinate residue (Tyr 385) contributes an election to compound I producing an oxyferryl heme and a tyrosyl radical (intermediate II). Finally, the tyrosyl radical abstracts a hydrogen from AA to begin the COX cycle of oxygen insertion and cyclization reactions. Neither the identity nor the source of the hydroperoxide necessary to initiate the first heme oxidation in vivo is known. In vitro, there is typically sufficient hydroperoxide in commercial fatty acid substrate preparations to initiate the process, and, once started, a hydroperoxide (e.g. PGG2 ) becomes available to continue the process as necessary. The POX reaction requires a reducing cosubstrate to convert compound I to II, and compound II to the heme of the resting enzyme. The identity of the reducing cosubstrate, in vivo, is not known. Interestingly, once the COX catalytic cycle has been initiated, it can operate independently of the POX cycle [8]. That is, the POX and COX reactions are not tightly coupled in the sense that there is a one to one correspondence between peroxide reduction and PGG2 formation [9]. Viewed from another perspective, the oxyferryl heme group of intermediate II, which is the same as the oxyferryl heme group of compound II of the POX cycle, can be reduced by one electron coming from a reducing cosubstrate to yield resting heme (Fe3+ -protoporphyrin IX) while the COX cycle continues to function [8].
3. PGHS peroxidase catalysis Although COX catalysis requires an initial oxidation of the heme group at the POX active site, POX catalysis can operate at or near maximal efficiency in the absence of COX turnover or occupancy of the COX active site [8,10,11]. PGHS POX has many of the same catalytic and spectroscopic properties of well-studied POXs including cytochrome c and horseradish POXs [12]. As noted by Garavito and coworkers [13] in the pioneering work on PGHS crystallography, the structure of PGHS-1 is closely related to that of myeloperoxidase; and, not unexpectedly, the structure of PGHS-2 is very similar to that of PGHS-1 [14,15]. POX rates are conveniently measured spectroscopically by monitoring changes in the absorbance of electron donors as they reduce oxidized heme intermediates formed when a hydroperoxide reacts with the heme group of the enzyme (Fig. 2A). N,N,N ,N -tetramethylphenylenediamine (TMPD) [16] and guaiacol [17] are the electron donors that have been used most widely in studying PGHS POX activities. These latter spectroscopic protocols
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Fig. 2. Interrelationships between COX and POX. (A) Mechanistic interrelationships between the COX and POX catalytic cycles. (B) Spatial relationships among catalytically important residues in COX and POX catalysis. AA, arachidonic acid; PPIX, protoporphorin IX; ROOH, an alkyl hydroperoxide (e.g. PGG2 ); ROH, an alkyl alcohol; I, compound I; II, compound II.
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measure the overall rate of the POX reaction. However, it is also possible to measure the rates of formation of the individual intermediates in POX catalysis (i.e. compounds I and II) using stopped flow spectrophotometry on a millisecond time scale. A characteristic of POXs is that they form spectroscopically identifiable oxidized heme intermediates during their catalytic cycles. As discussed before, the heme group at the POX active site of PGHS reacts with a hydroperoxide to form compound I [18–20]. Native PGHS-1 has a Soret peak for resting heme at 410 nm which is reduced in intensity and red shifted in forming compound I with an isosbestic point at 414 nm. This reaction, although rate limiting in POX catalysis, is actually relatively rapid with second order rate constants for hydroperoxide substrates in the range of 107 mol−1 s−1 ; in practical terms this means that when PGHS-1 is incubated with a small excess of a hydroperoxide substrate such as ethylhydrogen peroxide at concentrations in the millimolar range, compound I formation is complete within 10 ms at 4 ◦ C. In the presence of exogenous electron donors such as guaiacol or TMPD, compound I undergoes two very rapid, successive one-electron reductions, resulting in the formation of compound II and then resting enzyme (Fig. 2). PGHS-1 compound II has a prominent Soret peak at 424 nm [20]. Although Fe3+ -protoporphyrin IX is the natural heme ligand, Mn3+ -heme, but not other heme forms [21], will substitute in POX catalysis by PGHSs forming higher oxidation states of Mn3+ analogous to those seen with Fe3+ [22–24]. The specific activity of the Mn3+ -heme form of PGHS-1 is about 5% that of the Fe3+ -heme form.
4. Peroxidase substrate specificities The POX activities of PGHSs can reduce a variety of peroxides, but both isozymes, PGHS-1 and -2, show a preference for secondary alkyl hydroperoxides, among which is PGG2 , probably the physiologically most important substrate [20,25]. PGHS-1 POX activity has relatively low activity with tertiary hydroperoxides such as cumene hydroperoxide and t-butyl-hydroperoxide [26]. In vitro, relatively hydrophobic alkyl hydroperoxides such as 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP) exhibit about 10-fold higher secondary rate constants for formation of compound I than ethylhydroperoxide [20] about 1000-fold higher rate constants than observed with H2 O2 . Lower apparent Km values for the POX reaction as measured by rates of oxidation of reducing cosubstrates are also observed for relatively hydrophobic primary and secondary hydroperoxides (e.g. ∼10 versus 300 M for H2 O2 ) [20,25]. The PGHS POX reaction occurs at a heme-containing active site that appears in crystal structures to be near the protein surface and relatively exposed to solvent [13–15]. Therefore, the hydroperoxide substrate specificity is difficult to rationalize based on crystal structures. Moreover, while cyanide binds reasonably tightly to the heme group and in a linear orientation [27], the slightly larger azide group does not tightly (Seibold, unpublished data); this low affinity for azide is surprising because, again, there is no “ceiling” over the POX active site in the crystal structures of either PGHS-1 of PGHS-2. PGHS POX activity has relatively little specificity toward reducing cosubstrates [12,17,28]. However, two points of interest are that ascorbate is a poor reductant and that hydroperoxides themselves can function as reducing cosubstrates, which can cause problems in interpreting reaction kinetics [29].
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Residues thought to be involved in coordination of the heme iron or in POX catalysis have been examined by mutagenic analyses [25,27,30]. The heme irons of most POXs are coordinated by the four nitrogens of the protoporphyrin ring and at the fifth coordination position by the N␦ atoms of the imidazole group of the proximal histidine. In some cases, the iron is also coordinated at the sixth position either with a small inorganic ion or water. The classic push–pull hypothesis used to rationalize POX catalysis by heme POXs is that the distal histidine located near the sixth coordination position pulls a proton from the peroxide substrate as one of the oxygen atoms binds the sixth coordination position of the heme iron while a strongly basic, relatively electron-rich imidazolate anion of the proximal histidine ligand helps stabilize higher oxidation states of iron during oxidation of the heme [31]. Based on this hypothesis, the distal histidine of PGHS-1, His 207, would be predicted to be important in the deprotonation of the hydroperoxide substrate and subsequent protonation of the incipient alkoxide ion to form the alcohol during generation of compound I [25,30] and His 388, the proximal heme ligand, would be expected to have considerable anionic character. Additionally, Gln 203 would be considered to be important in facilitating heterolytic cleavage of the oxygen–oxygen bond by stabilizing the negative charge developing on the incipient alkoxide ion. Not surprisingly, results from site directed mutatgenesis indicate that indeed His 207, Gln 203 and His 388 are important in catalysis [25,27,30]. The results of studies of mutants of His 207 are consistent with its role as the distal histidine. However, the results of studies of mutants of Gln 203 and His 388 are not entirely consistent with their predicted roles in catalysis. Substitution of Gln 203 with arginine eliminates POX activity despite the fact that one might expect an arginine at this position to stabilize the alkoxide [25]; moreover, conversion of Gln 203 to an asparagine yields a mutant enzyme that is quite active although having a smaller residue at position 203 would be expected for steric reasons to be considerable less effective in charge stabilization. Not unexpectedly, H388Q and H388A mutants of PGHS-1 are inactive [30]. As noted before His 388 is the proximal (fifth) heme ligand with its N␦ imidazole nitrogen bonded to Fe3+ ; the Nε imidazole nitrogen may bond a water molecule that, in turn, may bond Tyr 504 [27]. In most heme POXs including myeloperoxidase, the proximal histidine interacts with an aspartate group causing the imidazole to be relatively basic (i.e. anionic), which increases the strength of the bond between the heme Fe3+ and the N␦ of the imidazole drawing the iron out of the heme plane and favoring a five-coordinate state. However, crystallographic data and visible and resonance Raman spectroscopy of resting oPGHS-1 indicate that it is mainly in the high-spin ferric form with Fe3+ in the plane of the ring and a water occupying the distal (sixth) coordination position [27]. Furthermore, resonance Raman spectroscopy of the complex between CO and the reduced enzyme (i.e. Fe2+ heme) indicate that imidazole group of His 388 is neutral, not anionic. Finally, substitution of Tyr 504 with alanine, a change that would disrupt any H2 O-mediated interaction between Tyr 504 and His 388, had little effect on PGHS-1 POX activity [27]. The generally accepted push–pull mechanism for POX catalysis requires strong basicity of the proximal ligand [32], His 388 in case of PGHSs. Collectively however, these recent studies on the heme–His 388 interaction in PGHS-1 imply that a strongly basic proximal ligand is not necessary for PGHS POX catalysis and that the “push” feature of the proposed mechanism for POX catalysis does not apply to PGHS-1 [27]. A neutral proximal histidine has also been observed two marine worm POXs [33].
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5. Comparisons of the peroxidase activities of PGHS-1 and -2 There are two significant differences between the POX activities of PGHS-1 and -2. First, PGHS-1 catalyzes primarily a standard two-electron reduction of hydroperoxide involving heterolytic cleavage of the peroxide group and yielding compound I and an alcohol; in contrast, PGHS-2 catalyzes 60% two-electron and 40% one-electron reductions [25]. The abilities of native and mutant PGHS-2 to catalyze heterolytic cleavage of peroxides correlate with their abilities to catalyze the COX reaction, which is consistent with compound I being the precursor of intermediate II (Fig. 2A) [25]. It is unclear if there is any biological significance to homolytic cleavage of hydroperoxides catalyzed by PGHS-2, and there is no obvious structural reason why PGHS-2 tends to produce close to 50% homolytic cleavage. A second significant difference between the POX activities of PGHS-1 and -2 is that the rate of formation of intermediate II formation from compound I is much faster with PGHS-2 [20]. This accounts in part for the fact that for PGHS-2, intermediate II is formed more rapidly and at lower peroxide concentrations. As a consequence of this more facile formation of intermediate II with its associated tyrosyl radical, PGHS-2 COX activity is activated at lower concentrations of hydroperoxide than those required to activate PGHS-1 [34,35]. This is potentially important biologically in that under conditions of low “peroxide tone” in cells, PGHS-2 could function in the presence of PGHS-1 without PGHS-1 being able to function [2,6,20]. Once again, there is no obvious structural explanation for the differences in rates of intermediate II formation property by PGHS-1 versus PGHS-2. 6. PGHS peroxidase inactivation PGHS POX activity is lost during reaction with various hydroperoxides. This inactivation process is a mechanism-based, suicide inactivation with t1/2 values in the range of 2–20 s depending on the reaction conditions [36,37]. The process occurs independent of the structure of the peroxide or occupancy of the COX active site [11,37,38]. Stopped flow spectroscopic and kinetic data originally suggested that POX inactivation involves a sequential conversion of compound I to COX intermediate II to III to a dead-end species with the slow step in the process being the last step-formation of the inactive enzyme [37,38]. More recently, however, it has become clear that the intermediate II in this process is actually an intermediate IIA with a tyrosyl radical being localized to a residue other than Tyr 385 (Fig. 2A) [11,39–41]. The nature of the protein modification(s) associated with suicide inactivation has not been defined. 7. Future directions in peroxidase research The POX activity of PGHS has not been studied in nearly so much detail as the COX activity of the enzyme so numerous questions remain. Two key areas of investigation focus on identifying the intermediates involved in the formation of both compound I and intermediate II and the basis for the unusual oxidant substrate specificity of PGHS POX. Studies in various laboratories are using molecular modeling, crystallographic, spectroscopic, and mutagenic approaches to address these questions.
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8. Cyclooxygenase catalysis The initial and rate limiting step in the COX reaction is the stereospecific removal of the 13-proS hydrogen from AA which yields an AA radical [42–44]. As depicted in Fig. 2B, an AA molecule becomes oriented in the COX active site with C-13 neighboring Tyr 385. Subsequent crystallographic analyses of AA in the active site of PGHS-1, that are described in further detail elsewhere in this compendium, indicate that the distance between the phenolic oxygen of Tyr 385 and the 13-proS hydrogen is about 2.5 Å [45]. This is very important because it indicates that AA is in a catalytically competent orientation (i.e. one that is converted to an oxygenated product) in the AA/PGHS-1 crystal. The AA radical formed in the rate limiting step has an unpaired electron centered initially on C-13. This radical isomerizes and the O2 diradical reacts with the AA radical having the electron centered at C-11 to yield the 11R-hydroperoxyl radical. In solution, a double allylic, carbon-centered radical formed at C-13 would rearrange to provide three major sites for O2 insertion at C-11, -13, and -15, yet greater than 97% of the O2 addition occurs at C-11 [46,47]. It is uncertain why this is so. It may be principally steric. There is more space for an O2 molecule under C-11 than near C-13 or -15 in the AA/PGHS-1 complex [45]. Alternatively or in addition, the strain existing in the conformation of the AA at the moment of hydrogen abstraction may cause the highest spin density to occur at C-11.1 Hydrogen abstraction and O2 insertion at C-11 do not require any change in the structure of AA [45]. However, formation of the carbon–carbon bond between C-8 and -12 requires that these atoms come within 1.5 Å, and in the AA crystal structure the distance between C-8 and -12 is about 4.5 Å. We have proposed that this occurs by rotation about the C-10/C-11 bond with the C-11 hydroperoxyl radical then adding to the back (top) side of C-9 to form the endoperoxide concomitant with movement of C-12 toward C-8 to form the cyclopentane ring [45]. Support for this concept comes from a crystal structure of the endoperoxide analog U44619 in the PGHS-1 COX site that shows from C-1 to -7 of this analog located in approximately the same positions as C-1 to -7 of AA and the hydroxyl group at C-15 of the analog located adjacent to Tyr 385.2 In fact, this latter observation suggests (a) that C-15 does move nearer the Tyr 385 position presumably favoring the second O2 addition at C-15, and (b) that in the PGG2 radical having the 15-hydroperoxyl radical substituent, that the 15-hydroperoxyl group can reabstract the hydrogen from Tyr 385 thereby regenerating the tyrosyl radical and yielding the PGG2 product. The 15-hydroperoxyl group of PGG2 is subsequently reduced to PGH2 by the POX activity of PGHSs. Based on the crystal structure, there is no direct route for PGG2 to travel through the protein from the COX to the POX site. Presumably, PGG2 exits the COX site through the opening in the membrane binding domain and travels around the surface of the protein to its site of reduction. 9. Cyclooxygenase substrate specificity PGHS-1 appears to use fatty acids such as arachidonate exclusively as substrates whereas PGHS-2 utilizes both fatty acids and 2-AG about equally well [48,49]. The ability of PGHS-2 1 2
Porter, personal communication. Malkowski, unpublished observation.
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to utilize alternative substrates may explain why there are two isozymes [6,48,49]; that is, one enzyme can generate a group of products that cannot be generated via the other isoform [50]. Arachidonate is the preferred fatty acid substrate for both PGHS-1 and -2. However, both enzymes will also oxygenate various n−3 and n−6 C18 and C20 fatty acids in vitro with catalytic efficiencies in the range of 0.05–0.7 of that of arachidonate [51]. At least some of these substrates including 9,12-octadecadienoate (18:2n−6), 8,11,14-eicosatrienoate (20:3n−6) and 5,8,11,14,17-eicosapentaenoate (20:5n−3) are oxygenated via COX activity when added exogenously to intact cells [52,53] or when mobilized from cellular phosphoglycerides [54–58]. In vesicular gland which has low levels of arachidonate, homo-␥-linolenic acid (20:3n−6) is converted efficiently to 1-series prostanoid products found in abundance in semen [56]. Other fatty acids that are COX substrates include adrenic acid (22:4n−6) [59], the Mead acid 5Z,8Z,11Z-eicosatrienoic acid [60], columbinic acid (5E,9Z,12Z-octadecatrienoic acid) [61,62], and 5,6-oxido-eicosatrienoic acid [63,64]. Substrates other than arachidonate typically have somewhat lower Km values than arachidonate but can compete with arachidonate for the COX active site thereby inhibiting formation of 2-series prostanoids [65]. This inhibition may be important in vivo in regulating overall prostanoid formation [66–68].
10. Multiple catalytically competent forms of substrates Closely related to the overall issue of substrate specificity is a subtle issue of the existence of different catalytically competent forms of the same substrate. PGHS-1 and -2 are both capable of forming more than one product from AA [46,69] and other substrate fatty acids [42]. PGHS-1 produces PGG2 , 11R-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid (11R-HETE), 15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15S-HETE) and 15R-HETE from AA. At saturating substrate concentrations about 95% of the product is PGG2 . Thus, there are obvious differences in the Vmax values for the formation of these products. Additionally, there are differences in the Km values (e.g. 5.5, 12, and 19 M for PGG2 , 11-HETE and 15-HETE formation, respectively) [46]. However, the KI values for inhibition of the formation of these different products by a competitive inhibitor are the same. These data are most easily interpretable as arachidonate being able to assume at least three catalytically productive arrangements within the COX site of PGHS-1.
11. Cyclooxygenase kinetics and their measurement There are three general methods for quantifying COX activity: (a) an O2 electrode assay for measuring O2 consumption involved in the conversion of arachidonate to PGG2 , (b) a radio-thin layer chromatography assay for measuring the conversion of 14 C-arachidonate to prostanoids, and (c) a sensitive luminescence assay which depends on the coordinate actions of COX and POX activities [70]. Note that for accurate measurements of O2 consumption corrections must be made for damping of electrode responses [9,11] and for suicide inactivation and peroxide activation [36,71].
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12. Cyclooxygenase inhibition by non-steroidal anti-inflammatory drugs PGHS-1 and -2 are the major pharmacological targets of non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs are competitive COX inhibitors which interfere with fatty acid substrate binding. Classical NSAIDs such as ibuprofen and naproxen inhibit the COX activity of both PGHS-1 and -2. The newly developed rofecoxib and celecoxib are more selective for PGHS-2. These latter drugs were developed to be anti-inflammatory and analgesic without causing the gastrointestinal side effects of classical NSAIDs. The structural basis for NSAID actions have been reviewed in detail elsewhere [3,5]. The most notable points at the enzymological level are the structural differences between the COX sites of the two isozymes that have permitted the development of isoform selective inhibitors [14,15] and the somewhat unusual pharmacological profiles that have been observed for the interactions the various drugs with the COXs [3,5,72]. Most notably, the new COX-2 inhibitors exhibit PGHS-2 selectivity because they inhibit this isoform by a time-dependent, pseudo-irreversible mechanism, while they inhibit PGHS-1 by a rapid, competitive, and reversible mechanism [3,5,72]. A detailed treatment of the topic of NSAIDs and a discussion of the mechanism of aspirin inhibition can be found elsewhere [3,5,72].
13. Cyclooxygenase inactivation Both the COX and POX activities of PGHS are inactivated during catalysis as the result of a non-productive breakdown of active enzyme intermediates. One COX active site is lost per about 2000 catalytic turnovers. The chemical changes in the protein that accompany these two suicide inactivation processes are unknown. The changes presumably involve reaction of amino acid radicals (e.g. tyrosyl radicals) with other groups on the protein and then reaction with molecular oxygen. Suicide inactivation is a crude regulatory mechanism which places an upper limit on cellular PG biosynthetic activity. It is unlikely that the intermediate(s) responsible for COX inactivation are substrate-derived radicals because the rate of covalent attachment of fatty acid to PGHS during catalysis is 30 times slower than that of suicide inactivation [73,74]. Interestingly, a Y385F oPGHS-1 mutant undergoes POX suicide inactivation at approximately the same rate as native PGHS-1 [11]; additionally, more than half of the POX activity can be retained following complete loss of COX activity under certain conditions [11]. COX inactivation involves COX intermediate II whereas POX inactivation occurs via intermediate IIA (Fig. 2A).
14. Future directions in cyclooxygenase research Three areas that are likely to be targets for future studies of COX activity are (a) chemical characterization of the process of COX suicide inactivation, (b) determination of the mechanism of formation of intermediate II from compound I, and (c) identification of the catalytically competent forms of AA that yield the 11R-HETE and 15R/S-HETE, respectively.
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Acknowledgements Work in the authors’ laboratories that was mentioned in this review was supported by NIH Grants DK22042 and GM57323. References [1] Marnett LJ, Rowlinson SW, Goodwin DC, Kalgutkar AS, Lanzo CA. Arachidonic acid oxygenation by COX-1 and COX-2. J Biol Chem 1999;274:22903–6. [2] Kulmacz RJ. Cellular regulation of prostaglandin H synthase catalysis. FEBS Lett 1998;430:154–7. [3] Smith WL, DeWitt DL, Garavito RM. Cyclooxygenases: structural, cellular and molecular biology. Annu Rev Biochem 2000;69:149–82. [4] DeWitt DL. Cox-2-selective inhibitors: the new super aspirins. Mol Pharmacol 1999;55:625–31. [5] Marnett LJ, Kalgutkar AS. Cyclooxygenase 2 inhibitors: discovery, selectivity and the future. Trends Pharmacol Sci 1999;20:465–9. [6] Smith WL, Langenbach R. Why there are two cyclooxygenases? J Clin Invest 2001;107:1491–5. [7] Dietz R, Nastainczyk W, Ruf HH. Higher oxidation states of prostaglandin H synthase. Rapid electronic spectroscopy detected two spectral intermediates during the peroxidase reaction with prostaglandin G2. Eur J Biochem 1988;171:321–8. [8] Koshkin V, Dunford HB. Coupling of the peroxidase and cyclooxygenase reactions of prostaglandin H synthase. Biochem Biophys Acta 1999;1430:341–8. [9] Wei C, Kulmacz RJ, Tsai AL. Comparison of branched-chain and tightly coupled reaction mechanisms for prostaglandin H synthase. Biochemistry 1995;34:8499–512. [10] Mizuno K, Yamamoto S, Lands WEM. Effects of non-steroidal anti-inflammatory drugs on fatty acid cyclooxygenase and prostaglandin hydroperoxidase activities. Prostaglandins 1982;23:743–57. [11] Song I, Ball TM, Smith WL. Different suicide inactivation processes for the peroxidase and cyclooxygenase activities of prostaglandin endoperoxide H synthase-1. Biochem Biophys Res Commun, in press. [12] Smith WL, Marnett LJ. Prostaglandin endoperoxide synthases. In: Sigel H, Sigel A, editors. Metal ions in biological systems, vol. 30. New York: Marcel Dekker, 1994. p. 163–99. [13] Picot D, Loll PJ, Garavito M. The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. Nature 1994;367:243–9. [14] Luong C, Miler A, Barnett J, Chow J, Ramesha C, Browner MF. Flexibility of the NSAID binding site in the structure of human cylcooxygenase-2. Nat Struct Biol 1996;3:927–33. [15] Kurumbail RG, Stevens AM, Gierse JK, McDonald JJ, Stegeman RA, Pak JY, et al. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nature 1996;384:644–8. [16] Kulmacz RJ. Prostaglandin G2 levels during reaction of prostaglandin H synthase with arachidonic acid. Prostaglandins 1987;34:225–40. [17] Markey CM, Alward A, Weller PE, Marnett LJ. Quantitative studies of hydroperoxide reduction by prostaglandin H synthase. Reducing substrate specificity and the relationship of peroxidase to cyclooxygenase activities. J Biol Chem 1987;262:6266–79. [18] Lambeir AM, Markey CM, Dunford HB, Marnett LJ. Spectral properties of the higher oxidation states of prostaglandin H synthase. J Biol Chem 1985;260:14894–6. [19] Karthein R, Dietz R, Nastainczyk W, Ruf HH. Higher oxidation states of prostaglandin H synthase. EPR study of a transient tyrosyl radical in the enzyme during the peroxidase reaction. Eur J Biochem 1988;171:313–20. [20] Lu G, Tsai A-L, Van Wart HE, Kulmacz RJ. Comparison of the peroxidase reaction kinetics of prostaglandin H synthase-1 and -2. J Biol Chem 1999;274:16162–7. [21] Ogino N, Ohki S, Yamamoto S, Hayaishi O. Prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. Inactivation and activation by heme and other metalloporphyrins. J Biol Chem 1978;253:5061–8. [22] Tsai A, Wei C, Baek HK, Kulmacz RJ, Van Wart HE. Comparison of peroxidase reaction mechanisms of prostaglandin H synthase-1 containing heme and mangano protoporphyrin IX. J Biol Chem 1997;272: 8885–94.
126
W.L. Smith, I. Song / Prostaglandins & other Lipid Mediators 68–69 (2002) 115–128
[23] Odenwaller R, Maddipati KR, Marnett LJ. Detection of a higher oxidation state of manganese-prostaglandin endoperoxide synthase. J Biol Chem 1992;267:13863–9. [24] Strieder S, Schaible K, Scherer HJ, Dietz R, Ruf HH. Prostaglandin endoperoxide synthase substituted with manganese protoporphyrin IX. Formation of a higher oxidation state and its relation to cyclooxygenase reaction. J Biol Chem 1992;267:13870–8. [25] Landino LM, Crews BC, Gierse JK, Hauser SC, Marnett LJ. Mutational analysis of the role of the distal histidine and glutamine residues of prostaglandin-endoperoxide synthase-2 in peroxidase catalysis, hydroperoxide reduction, and cyclooxygenase activation. J Biol Chem 1997;272:21565–74. [26] Ohki S, Ogino N, Yamamoto S, Hayaishi O. Prostaglandin hydroperoxidase, an integral part of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J Biol Chem 1979;254:829–36. [27] Seibold SA, Cerda JF, Mulichak AM, Song I, Garavito RM, Arakawa T, Smith WL, Babcock GT. Peroxidase activity in prostaglandin endoperoxide H synthase-1 occurs with a neutral histidine proximal heme ligand. Biochemistry 2000;39:6616–24. [28] Hsuanyu Y, Dunford HB. Prostaglandin H synthase kinetics: the effect of substituted phenols on cyclooxygenase activity and the substituent effect on phenolic peroxidatic activity. J Biol Chem 1992;267:17649–57. [29] Hsuanyu Y, Dunford HB. Kinetics of the reaction of prostaglandin H synthase compound II with ascorbic acid. Arch Biochem Biophys 1990;281:282–6. [30] Shimokawa T, Smith WL. Essential histidines of prostaglandin endoperoxide synthase. J Biol Chem 1991;266:6168–73. [31] Poulos TL, Kraut J. The stereochemistry of peroxidase catalysis. J Biol Chem 1980;255:8199–205. [32] Dawson JH. Probing structure-function relations in heme-containing oxygenases and peroxidases. Science 1988;240:433–9. [33] Franzen S, Roach MP, Chen YP, Dyer B, Woodruff WH, Dawson JH. The unusual reactivities of Amphitrite ornata dehaloperoxidase and Notomastus lobatus chloroperoxidase do not arise from a histidine imidazolate proximal heme iron ligand. J Am Chem Soc 1998;8120:4658–61. [34] Capdevila JH, Morrow JD, Belosludtsev YY, Beauchamp DR, DuBois RN, Falck JR. The catalytic outcomes of the constitutive and the mitogen inducible isoforms of prostaglandin H2 synthase are markedly affected by glutathione and glutathione peroxidase(s). Biochemistry 1995;34:3325–37. [35] Kulmacz RJ, Wang LH. Comparison of hydroperoxide initiator requirements for the cyclooxygenase activities of prostaglandin H synthase-1 and -2. J Biol Chem 1995;270:24019–23. [36] Callan OH, So OY, Swinney DC. The kinetic factors that determine the affinity and selectivity for slow binding inhibition of human prostaglandin H synthase 1 and 2 by indomethacin and flurbiprofen. J Biol Chem 1996;271:3548–54. [37] Wu G, Wei C, Kulmacz RJ, Osawa Y, Tsai A-L. A mechanistic study of self-inactivation of the peroxidase activity in prostaglandin H synthase-1. J Biol Chem 1999;274:9231–7. [38] Wu G, Vuletich JL, Kulmacz RJ, Osawa Y, Tsai A-L. Peroxidase self-inactivation in prostaglandin H synthase-1 pretreated with cyclooxygenase inhibitors or substituted with mangano protoporphyrin IX. J Biol Chem 2001;276:19879–88. [39] Tsai A, Hsi LC, Kulmacz RJ, Palmer G, Smith WL. Characterization of the tyrosyl radicals in ovine prostaglandin H synthase-1 by isotope replacement and site-directed mutagenesis. J Biol Chem 1994;269:5085–91. [40] Hsi LC, Hoganson CW, Babcock GT, Garavito RM, Smith WL. An examination of the source of the tyrosyl radical in ovine prostaglandin endoperoxide synthase-1. Biochem Biophys Res Commun 1995;207:652–60. [41] Shi W, Hoganson CW, Espe M, Bender CJ, Babcock GT, Palmer G, et al. Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1. Biochemistry 2000;39:4112–21. [42] Hamberg M, Samuelsson B. On the mechanism of the biosynthesis of prostaglandins E-1 and F-1-alpha. J Biol Chem 1967;242:5336–43. [43] Tsai A, Kulmacz RJ, Palmer G. Spectroscopic evidence for reaction of prostaglandin H synthase-1 tyrosyl radical with arachidonic acid. J Biol Chem 1995;270:10503–8. [44] Tsai A, Palmer G, Xiao G, Swinney DC, Kulmacz RJ. Structural characterization of arachidonyl radicals formed by prostaglandin H synthase-2 and prostaglandin H synthase-1 reconstituted with mangano protoporphyrin IX. J Biol Chem 1998;273:3888–94.
W.L. Smith, I. Song / Prostaglandins & other Lipid Mediators 68–69 (2002) 115–128
127
[45] Malkowski MG, Ginell S, Smith WL, Garavito RM. The X-ray crystal structure of prostaglandin endoperoxide H synthase-1 complexed with arachidonic acid. Science 2000;289:1933–7. [46] Thuresson ED, Lakkides KM, Smith WL. Different catalytically competent arrangements of arachidonic acid within the cyclooxygenase active site of prostaglandin endoperoxide H synthase-1 lead to the formation of different oxygenated products. J Biol Chem 2000;275:8501–7. [47] Thuresson ED, Lakkides KM, Rieke CJ, Sun Y, Wingerd BA, Micielli R, et al. Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid. J Biol Chem 2001;276:10347–57. [48] Kozak KR, Rowlinson SW, Marnett LJ. Oxygenation of the endocannabinoid, 2-arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2. J Biol Chem 2000;275:33744–9. [49] Kozak KR, Prusakiewicz JJ, Rowlinson SW, Schneider C, Marnett LJ. Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid 2-arachidonylglycerol. J Biol Chem 2001;276: 30072–7. [50] Kozak KR, Crews BC, Ray JL, Tai H-H, Morrow JD, Marnett LJ. Metabolism of prostaglandin glycerol esters and prostaglandin ethanolamides in vitro and in vivo. J Biol Chem 2001;276:30072–7. [51] Laneuville OI, Breuer DK, Xu N, Huang ZH, Gage DA, Watson JT, et al. Fatty acid substrate specificities of human prostaglandin H synthases-1 and -2: formation of 12 hydroxy-(9Z,13E/Z,15Z)-octadecatrienoic acids from alpha-linolenic acid. J Biol Chem 1995;270:19330–6. [52] Baer AN, Costello PB, Green FA. Stereospecificity of the hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids produced by cultured bovine endothelial cells. Biochim Biophys Acta 1991;1085: 45–52. [53] Kaduce TL, Figard PH, Leifur R, Spector AA. Formation of 9-hydroxyoctadecadienoic acid from linoleic acid in endothelial cells. J Biol Chem 1989;264:6823–30. [54] Baer AN, Costello PB, Green FA. Stereospecificity of the products of the fatty acid oxygenases derived from psoriatic scales. J Lipid Res 1991;32:341–7. [55] Abeywardena MY, Fischer S, Schweer H, Charnock JS. In vivo formation of metabolites of prostaglandins I2 and I3 in the marmoset monkey (Callithrix jacchus) following dietary supplementation with tuna fish oil. Biochim Biophys Acta 1989;1003:161–6. [56] Knapp HR. Prostaglandins in human semen during fish oil ingestion: evidence for in vivo cyclooxygenase inhibition and appearance of novel trienoic compounds. Prostaglandins 1990;39:407–23. [57] Leaver HA, Howie A, Wilson NH. The biosynthesis of the 3-series prostaglandins in rat uterus after alpha-linolenic acid feeding: mass spectroscopy of prostaglandins E and F produced by rat uteri in tissue culture. Prostaglandins Leukot Essent Fatty Acids 1991;42:217–24. [58] Engels F, Willems H, Nijkamp FP. Cyclooxygenase-catalyzed formation of 9-hydroxylinoleic acid by guinea pig alveolar macrophages under non-stimulated conditions. FEBS Lett 1986;209:249–53. [59] Larsen LN, Dahl E, Bremer J. Peroxidative oxidation of leuco-dichlorofluorescein by prostaglandin H synthase in prostaglandin biosynthesis from polyunsaturated fatty acids. Biochim Biophys Acta 1996;1299:47–53. [60] Oliw EH, Hornsten L, Sprecher H, Hamberg M. Oxygenation of 5,8,11-eicosatrienoic acid by prostaglandin endoperoxide synthase and by cytochrome P450 monooxygenase: structure and mechanism of formation of major metabolites. Arch Biochem Biophys 1993;305:288–97. [61] Elliott WJ, Morrison AR, Sprecher HW, Needleman P. The metabolic transformations of columbinic acid and the effect of topical application of the major metabolites on rat skin. J Biol Chem 1985;260:987–92. [62] Nugteren DH, Christ Hazelhof E. Naturally occurring conjugated octadecatrienoic acids are strong inhibitors of prostaglandin biosynthesis. Prostaglandins 1987;33:403–17. [63] Oliw EH. Metabolism of 5(6)oxidoeicosatrienoic acid by ram seminal vesicles. Formation of two stereoisomers of 5-hydroxyprostaglandin I1. J Biol Chem 1984;259:2716–21. [64] Balazy M. Metabolism of 5,6-epoxyeicosatrienoic acid by the human platelet. Formation of novel thromboxane analogs. J Biol Chem 1991;266:23561–7. [65] Lands WEM, LeTellier PR, Rome LH, Vanderhoek JY. Inhibition of prostaglandin biosynthesis. Adv Biosci 1973;9:15–28. [66] Needleman P, Whitaker MO, Wyche A, Watters K, Sprecher H, Raz A. Manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: pharmacological basis for a therapeutic approach. Prostaglandins 1980;19:165–81.
128
W.L. Smith, I. Song / Prostaglandins & other Lipid Mediators 68–69 (2002) 115–128
[67] Spector AA, Kaduce TL, Figard PH, Norton KC, Hoak JC, Czervionke RL. Eicosapentaenoic acid and prostacyclin production by cultured human endothelial cells. J Lipid Res 1983;24:1595–604. [68] Lagarde M, Drouot B, Guichardant M, Dechavanne M. Uptake and effect on arachidonic acid oxygenation of some icosaenoic acids in human platelets. Biomed Biochim Acta 1984;43:S319–22. [69] Xiao G, Tsai AL, Palmer G, Boyar WC, Marshall PJ, Kulmacz RJ. Analysis of hydroperoxide-induced tyrosyl radicals and lipoxygenase activity in aspirin-treated human prostaglandin H synthase-2. Biochemistry 1997;36:1836–45. [70] Laneuville O, Manke CR, DeWitt DL, Smith WL. Assays for cyclooxygenase inhibiton by nonsteroidal anti-inflammatory drugs. In: Allison AC, editor. Weir’s handbook of experimental immunology, vol. IV. 5th ed. Oxford, UK: Blackwell 1995. p. 1–6. [71] Chen W, Pawelek TR, Kulmacz RJ. Hydroperoxide dependence and cooperative cyclooxygenase kinetics in prostaglandin H synthase-1 and -2. J Biol Chem 1999;274:20301–6. [72] Smith WL, DeWitt DL. Prostaglandin endoperoxide H synthases-1 and -2. In: Dixon FJ, editor. Advances in immunology, vol. 62. San Diego, CA: Academic Press 1996. p. 167–215. [73] Kulmacz RJ. Attachment of substrate metabolite to prostaglandin H synthase upon reaction with arachidonic acid. Biochem Biophys Res Commun 1987;148:539–45. [74] Lecomte M, Lecocq R, Dumont JE, Boeynaems J-M. Covalent binding of arachidonic acid metabolites to human platelet proteins. J Biol Chem 1990;265:5178–87.