B,,chhmca et Biopl~vsi,'a Acta. 1()~3 (1991) 1 -17

' 1991 Elsevier Science Publishers B.V. 0005-2760/ql/$03.50 ADONIS (X)05276091001438

BBALIP 53619

Review

Prostaglandin endoperoxide synthase: structure and catalysis William L. Smith ~ and Lawrence J. Marnett

2

t Depurtnlent nf Bmc& mi.~tr)'. Mtcht.t¢an State Unwersm', East Lansing, MI r U . S . A I and : The ,4, R ttam'ock, Jr. Memorial Laboratory for Cancer Re.~earch. Department of Btv('hemistrv. Center in ,'~iolecular T~xzeologv. Vanderbdt Unit 'er.~'tQ'School of ,~4edlctne, ,¥asht'dk', 7 N ( U. S. A. )

(Received 31 August t990) (Revised manuscript received 20 December ITS90)

Key words: Cyclooxygenase: Peroxidase: Nonsteroidal anti-inflammatory drug: Aspirin: Hcmc: Co-oxidation

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

Overview of prostaglandin bk~hemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A, Prostanoid biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Prostanoid functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 2

Catalytic properties: cydoo~ygenase rear.lion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cyclooxygenase substrat,~ specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The cyclooxygenase reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C, Modulation et ,:ycl0or.y,~;enaseactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. 02 and fatty acid substrate concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E, Inhibition of cycloox)'gcltasc activity by rcdudnr, h)dmperoxidc a~fivator concentrations . . . . . F. Inhibition of cycloox,~',~ase activity by non,;eroidal anti-inflammatory drugs . . . . . . . . . . . . .

4 4 4 5 5

4

5

6

Catalytic properties: peroxidase reaclion ..........................................

i

A. Peroxide substratc specificity ................................................

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B. Spectral intermediates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Peroxidase reducing suhstratcs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7

Cyclooxygenase and peroxidase relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The 'suicide' inaclivaliou of cyclooxygt:nase versus peroxide-dependent inactivation of pcroxidas¢ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Interrelationship between cyck×)xygcnasc and peroxidase catalysis . . . . . . . . . . . . . . . . . . . . . VI. Struclural properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General properties of purtfi,;d PGI I symhas¢ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Glycosyladon sizes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Heine binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Active silt: tyrosin¢ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9

l0

I0 10 II

I1 12 13 13

Abbreviations: PG. prostaglandin; OSH, reduced glulathione; PPHP, 5-phenyl-4-pcntcnyl-l-hydropcroxide; Tx. Ihromhoxane; ADH. amidiuretic hormone; PI, phosphatidylino~itol; ETYA, 5.8,tl,14-eicosatetraynoic acid: Fc~'-PPIX, ffoniIII)protoporphyrin IX; Mn'~+-PPIX, manganesc(llI)protoporphyrin IX; 15.HPETE, I5(SFhydroperoxy-5cis,gc~s.l h,r~,I3tran.~'-cicosatdraenoic acid; EPR, electron paramagnetic resonance: SDS.PAGE, sodium dodccyl sulfate-poiyacrylamide gel electrophoresis; Man. mannose: OIcNAc, N-acctyl-glucosamine', EGF, epidermal growth factor: TNM, tetranitromethanc; cicosanoid is a general term for bioactive lipids derived from 20 carbon polyunsaturated fatty acids: prostanoid is a general term for bioactive lipids containing the prostanoic acid nucleus.

Correspondence: W.L. Smilh, Department of Biochemistry, Biochemistry Building, Michigan State University, East Lansing. MI 48824, U.SA

F.. Aspirin acety;ation site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. X-ray cryslalh)graphy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vll. Active site model for PGH synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Fulure :liras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. Introduction On an economic basis, it can be reasonably argued that prostaglandin endoperoxide (PGH) synthase is the world's most important enzyme because it is the target site of nonsteroidal anti-inflammatory drugs that account for 3-5 billion dollars in annual pharmaceutical and over-the-counter sales (e.g., aspirin, ibuprofen). With recent data indicating that low-dose aspirin also diminishes the incidence of cardiovascular disease, the regulation of this enzyme takes on even greater pharmacological importance. In this review, we describe the properties of PGH synthase, tile enzyme which catalyzes the committed step in the synthesis of prostaglandins and thromboxane. We begin by presenting an overview of protanoid biosynthesis and the mechanism of action of these local hormones. We then describe the novel characteristics of the two distinct reactions catalyzed by PGH synthase-the cyclooxygenase and peroxidase reactions-and the interdependence of these two transformations. Finally, we summarize the chemical and physical characteristics of the enzyme protein, insofar as possible relating structure to catalysis and we propose a model for the active site of PGH synthase. In an upcoming complementary review, DeWitt will address aspects of PGH synthase gene structure and the regulation of expression of the PGH synthase gene [1]. II. Overview of prostaglandin biochemistry I I-A. ProstanoM biosynthesis

Fig. 1 summarizes the three phases of prostanoid formation: (a) mobilization of arachidonic acid from cellular phosphoglycerides; (b) sequential conversion of 'released' arachidonate to the ptostagiandin cl~doperoxides PGG_, then PGH2: and (c) either isomerization or reduction ~f PGH, to what are considered to be biologically important dcriwltives (PGD,, PGE:, PGF.,,,), thromboxane A 2 (TxA.,) and prostacyclin (PGI2) [2,3]. Initiation of prostanoid synthesis occurs when a hormone or proteinase interacts with an appropriate receptor or proteinase target on the cell surface. Each stimulus appears to act in a cell-specific manner. For example, bradvkinin and antidiuretic hormone selectively activate PGE 2 synthesi~ in renal collecting tubule cells [45] and a-thrombin stimulates PGI2 synthesis by vascular endothelial cells [6], Interaction of a stimulus

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I ~14 14 15

with a target cell leads to activation of one or more lipase systems. The two lipase systems implicated as being important in prostanoid formation are: (a) phospholipase A~ acting on phospbatidylethanolamine, phosphatidylcholine, or plasmalogens; and (b) phospholipase C and diacylglyeerol lipase acting sequentially on phosphatidylinositol derivatives (see Ref. 7 for review). Arachidonate is often mobilized via both pathways in stimulated cells [8,9]; the contribution of each pathway to arachidonate mobilization is still unclear although the current opinion is that the phospholipase A 2 pathway is the most important [7]. Once arachidonate is released, it can be acted upon by PGH synthase-tbe major subject of this review. This enzyme exhibits both a bis-oxygenase (cyclooxygenase) activity catalyzing PGG 2 formation and a peroxidase activity catalyzing a two-electron reduction of PGG z to PGH 2. Both cyclooxygenase and peroxidase activities are associated with a single protein molecule [10-12]. PGH synthase is an integral membrane protein found mainly in microsomal membranes [13-151. Subcellular localization studies have indicated that the enzyme is concentrated in the endoplasmic reticulum but that in the same cell PGH synthase is also found on the nuclear envelope and often on the plasma membrane [13,16,17] (Gerezissis, K. and Smith, W.L., unpublished data). It is unclear why the enzyme is associated with several different membrane systems; one possibility is that synthesis at different subcellular sites is elicited by different stimuli [13,14,16,181. The aspirin acetylation site [19], the site of trypsin cleavage [20] and the major antigenic determinants of POH synthase [19] are on the cytoplasmic side of the endoplasmic reticulum indicting that PGH 2 is generated intracellularly as opposed to extracellularly. Most of the synthases which further metabolize PGH 2 also appear to be present on the endoplasmic reticulum [13,14,181. Formation of the biologically active eicosah~,ids from PGH~ occurs through the actions of a set of synthases called PGD synthase [21-23 !, PGE synthase [24,25], PGF synthase [26], PGI synthase [27,28] and TxA syntbase [29,30]. Conversion of PGH z to PGFz, involves a net two-electron reduction of PGH 2. All other products are formed via non-redox isomerization reactions. Although all of the prostanoids are shown in the generic cell illustrated in Fig. 1, most prostaglandin-forming cells produce only one of these products because of the predominance of a single PGH z metabolizing enzyme.

i/r

STIMULUS CELL MEMBRANE

v

~PHOSPHOLIIpID

>

A CO TV ISP AH TO O I LN O PH P IAFSES

~ o o

H

202/ARACHIDONIC

AC;~

/ CYCLOOXYGENASE) i'" "~COOH i~f ~ (H.

•\ ~

"'~"" ~

L.',

.,..

FGH SYNTHASE

PROSTACYCU N " . ,coow

V "-.f " v ~ . . . ~ 1 .

~GD

OH

~.

-

-~

V

PGI2 OH

,,~ .o"

PGF

PGE 2

6.

/ ~'"'"~"

6H HO

I ,~,~

~l"'~

MO

.,,,.

1.,

V "cooH

TxA2

CH PGF2=

Fig. I. Biosyn|hctic pathway for proxtanoid formation.

For instance, blood platelets which contain TxA synthase [29] normally form only TxA, and not other prostanoids [311. Prostaglandin formation occurs in all mammalian species [13], and there are reports of synthesis occurring in fish [32] and insects [33]. Presumably, synthesis is limited to animals. Plants, yeast and bacteria lack the appropriate polyunsaturated fatty acid precursors. In

the case of mammals, prostanoid synthesis occurs in most organs, but typically not in all cell types comprising an organ [13]. For example, in kidney only a third of the 15 different cell types synthesize prostanoid products [34]. PGI 2 synthesis occurs in all smooth muscle cells [16] and PGI z or PGE 2 formation occurs in virtually all vascular endothelial ceils [14 I. (The latter observations explain why l~rostagtandin synthesis is seen

with all tissues). With the exception of these generalizations, there is no way to predict whether a cell can form prostanoids based on histology or embryology [13]. However, once synthesis has been shown to occur in a cell type from one species, the same cell type can be expected to form prostanoids in other species [13].

II-B. Prostanoid functions Once a prostaglandin is formed, it exits the cell, probably via carrier-mediated transport [14,35], then interacts with receptors on the parent cell and/or on closely neighboring cells to modulate second messenger levels [2]. Thus, prostanoids have both autocrine and paracrine functions. Prostanoids can be thought of as local hormones that are formed in response to and coordinate the effects of circulating hormones. An example is the renal collecting tubule where PGE 2 is synthesized in response to antidiuretic hormone (ADH). Newly formed PGE 2 functions on collecting tubule cells and on neighboring thick ascending limb cells (which do not form prostanoids) to attenuate cAMP formation induced in each cell type by ADH; the net physiological effects of PGE2 are to inhibit in a coordinate fashion ADH-induced water reabsorption by the collecting tubule and ADH-induced NaCI reabsorption by the thick ascending limb [2.4]. Studies on the biochemical mechanisms of prostanoid actions indicate that prostanoids act through receptors which belong to the G protein-linked receptor family [2]. It is likely that there is a subfamily of receptors for each prostanoid. An example is PGE 2. There appear to be three pharmacologically and functionally distinct receptors for PGE~ which are functionally homolegous to the fl-, a2-, and aj-adrenergic receptors [36-40]; these three different PGE receptors operate through different G proteins to activate adenylate cyclase [37,38], inhibit adenylate cyclase [37-391 and activate Pl-specific phospholipase C [40], respectively, !11. Catalytic properties: eydooxygenase reaction

II I-A. Q'clooxvgenase substrate specific'iO, A variety of polyunsaturated fatty acids are substratcs for the oxygenase activity. Fatty acids containing at [cast three methylene-interrupted c/s-double bonds beginning at n-6 are converted to PGG derivatives [41,42]. The rates of oxygeeation vary considerably with the number of the carbons in the fatty acid and the presence of additional double bonds (e.g., near the methyl end of the fatty acid), The best substrates for the cydooxygenase reaction are 8,11,14-eicosatrienoic acid (di-homo-~'-Iinolenic acid) and 5,8,11,14-eicosatetraenoic acid (arachidonic acid) (Kin= 2-10 ~M, Vm,~ = 1400 nmol/nraoi enzyme [43,44]), In vivo, the most

common cyclooxygenase substrate is arachidonate. Unsaturated fatty acids containing two methylene-interrupted double bonds (e.g., 11,14-eicosadienoic acid [45] or 9,12-octadecadienoic acid) are oxygenated to monohydroxy fatty acids. Although hydropero ides are presumed to be intermediates in the production of these hydroxy acids, they have not actually been isolated. Lands et al. [46] demonstrated that n-3 and n-9 fatty acids having 18-22 carbons cause significant inhibition of enzyme activity. Eieosapentaenoic acid (20 : 5(n-3)) can serve as a substrate when the peroxide concentrations are elevated [47]; however, the activity of the cyclooxygenase toward eicosapentaenoic acid is less than 50% of that Observed with arachldonic acid, a fact that may account for part of the apparent anti-thrombogenic activity of fish oils, Another significant constituent of fish oils is docosahexaenoic acid (22: 6(n-3)), This fatty acid is a potent competitive inhibitor of the oxygenation of arachidonate with a K i of about 5/~M [48]. Typical of other competitive cyclooxygenase inhibitors, 22 : 6 does not affect the peroxidase activity of PGH synthase. 5.8,11,14-Eicosatetraynoic acid (ETYA) has long been known to inhibit cyclooxygenase activity [49,50]. Inhibition is irreversible following incubation of the enzyme in the presence but not in the absence of oxygen and a hydroperoxide activator [50]. it is thought that ETYA acts as a suicide substrate (see below) which, once activated, reacts at the active site of the enzyme to prevent further access of other substrates. ETYA also irreversibly inhibits a variety of lipoxygenases (fatty acid dioxygenases) apparently by oxidizing an i~portant methionine residue to a methionine sutfoxide

i511. III.B. The cyciooxygenase reaction Fig. 2 is an adaptation of the mechanism of the cyclooxygenase reaction proposed by Hamberg and Samudsson in 1967 [52]. A considerable body of experimental evidence supports their proposed mechanism (reviewed in Ref. 3), In this model, PGH synthase holds the substrate molecule in a conformation in which a kink is present by virtue of rotation about the C-9/C-10 single bond [53]. In the first step. removal of the" pro-S hydrogen atom produces a carbon radical that is trapped by 02 at C-II. If one assumes that H removal occurs proximal to the protein molecule, it seems likely that 02 attacks C-11 from the side of the substrate exposed to the aqueous phase. This is on the opposite side of the fatty acid molecule from which the H was removed. Serial cyclization of this 1 l-peroxyl radical produces a bicyclic peroxide with trans aliphatic side chains, Attack of the second molecule of 02 on the carbon radical at C-15 should come from the same side of the fatty acid substrate as attack at CA1. Reduction of the

C02H

O~H

C02H

maximal activation of the cyclooxygenase activity of purified PGH synthase occurs with approx. 20 nM 15-HPETE [58] which is well below the estimated K m for the peroxidase activity of PGH synthase (approx. 10 t~M) [59]. Despite this concentration differential, there is a correlation between the ability of various peroxides to serve as cyclooxygenase activators and peroxidase substrates. In summary, hydroperoxide appears to be necessary for oxidation of the cyclooxygenase to a form which is capable of abstracting the 13-pro-S hydrogen from arachidonate. Later in the review, in discussing the interrelationship between the cyclooxygenase and peroxidase activities of PGH synthase, we will discuss a mechanism for hydroperoxide activation of the enzyme. III-C Modulation of (yclru~xygenase actiri O'

Fig. 2. Putativemechanismof arachidonicacid oxygenationby PGH synthase.AdaptedfromRcf.42.

resultant peroxyl radical produces PGG., with the stereochemistry of the natural product. Although the overall chemical transformation catalyzed by cyclooxygenase is complex, the function of the enzyme depicted in Fig. 2 is rather simple-fixation of the conformation of the substrate and stereospecific hydrogen removal. There is considerable similarity between the steps in the cyclooxygenase reaction and non-enzymatic fatty acid autoxidation. In fact, in 1966, Nugteren et al. [54 I reported the isolation of prostaglandin-like substances from autoxidized eicosatrienoic acid. Subsequent investigations revealed that the stereochemistry of attachment of the side chains to the five-membered ring is cis in contrast to the trans stereochemistry of naturally occurring prostaglandins. Two central questions about cyclooxygenase catalysis are the identity of the activated functional group on the enzyme that removes the 13-pro.S hydrogen and the way in which the oxidizing agent is generated. There is evidence that the initial activation requires an exogenous peroxide. Cyclooxygenase activity was shown some 20 years ago to be blocked by reduced glutathione (GSH) in the presence of excess GSH peroxidase [55]. This inhibition could be overcome by adding N-ethylmaleimide or peroxide. PGH synthase is also inhibited by cyanide at concentrations that bind to the heme prosthetic group. The lag phase in cyclooxygenase activity induced by cyanide can be overcome by low concentrations of fatty acid hydroperoxides [56,57]. Half-

In principle, the cyclooxygenase activity of PGH synthase can be inhibited by agents or maneuvers which: ~a) reduce the concentrations of either oxygen or fatty acid substrate; {b) reduce the concentrations of hydrop~:roxide n'eded for initiation; (c) reduce oxidized enzyme forms to the native form: or (d) interfere with arachidonate binding to the cyclooxygenase active site. In addition, anti-inflammatory steroids diminish cyclooxygenase activity (and peroxidase activity), apparently by inhibiting translation of the mRNA for PGH synthase [I.60,61 I. III.D. O: and fatty acid substrate c'omz,ntrations The K,, for O, for the cyclooxygenase reaction is about 5 p.M [621. Even in tissues bathed with blood having low oxygen concentrations, O: concentrations are greater than 20 pM. Hence, O, concentrations do not pose a constraint on the rate of the cyclooxygenase reaction in vivo except in anoxic tissue. The concentration of unesterified arachidonate in tissues is approx. 20 t.tM [43,63,64] which is well above the K., of the cyclooxygenase for arachidonate. However, this unesterified arachidonate must be in a form inaccessible to the cyclooxygenase because prostaglandin endoperoxide formation in intact cells only occurs following stimulation of phospholip,:~e activity [71. liI-E. Inhibition of cycloo.~.vgenase activity by reducing hydroperoxide activator concentrations As noted above, the cyelooxygenase has an obligatory requirement for a hydroperoxide 'activator'. Any maneuver which reduces hydroperoxide concentrations below about 10 uM can inhibit cyclooxygenase activity. Addition of GSH peroxidase in the presence of GSH causes inhibition of cyclooxygenase activity in vitro [43,551 and this inhibitory effect is potentiated by per-

o×idase electron donors [43]. Marshall et al. [43] have provided evidence that factors present in the cytosol of tissues such as ram seminal vesicles will potentiate the inhibitory effect of exogenous GSH peroxidase; moreover, they have reported that many cells contain levels of GSH and GSH peroxidase that will inhibit cyctooxygcnase activity. These findings suggest that there may be constraints on cyclooxygenase activity in intact cells that are normally not considered when studying the enzyme in vitro.

Aspirin E --,~

'--"

CO=H

Ibt~rolen

~

~

~CO,H

Ru~iprolen

lll-E Inhibition of o'clooxygenase activity by nonsteroidal atrti-inflammatmT drugs PGH synthase appears to be the target site for the action of many nonsteroidal anti-inflammatory agents 165.661. In generaI, these drugs compete with arachidonate for binding to the cyclooxygenase active site of the enzyme, but do not inhibit the peroxidase activity of the enzyme [67,68]. Subsequent to competitive cyclooxygenase inhibition, several of these inhibitors cause secondary effects including either covalent (e.g., aspirin) or noncovalent (e.g.. indomethacin) alterations of PGH synthase structure which result in irreversible inactivation of cyclooxygenase but not peroxidase activity [68,691. Most nonsteroidal anti-inflammatory drugs bear no close structural resemblance to arachidonate (Fig. 3). Many are related to one another having a (2S)-phenylpropionic acid structure (e.g., naproxen, clinoril, ibuprofen. flurbiprofen): however, meclofenamate, flurbiprofen, indomethacin, aspirin are only loosely related structurally. Flurbiprofen, meclofenamate, indomethacin, and aspirin cause irreversible inactivation of cyclooxygenase activity in vitro [69]. Of these agents, only aspirin causes an irreversible inactivation in vivo [70]. Aspirin functions in a two-step process. First, aspiri,t binds to the cyclooxygenase active site of PGH synthase competing with arachidonate for binding, albeit inefficiently, with a K, of about 20 mM [69,71]. Following binding, aspirin causes acetylation of the protein. The rate of acetylation is increa~,ed approx. 100-fold by the presence of the heine prosthetic group [72.73]. Roth et al. [741 de* termined that [3H]acetyisalicylate acetylates a single serine residue and sequenced a tryptic peptide containing the tritiated acetyl group. Subsequenl sequencing of the cDNAs coding for enzyme identified this residue as Ser ~3~ [75-77] *. Although these results suggested that the hydroxyl group o[ Ser ~~n might be important in

* The numbering system used for amino acid residues assigns the number I to the Met at the translational start signal deduced from

the eDNAsequence.In the case of the sheepenzyme,the initially translated protein is 24 residueskrugerthan the matureprotein.

CH30~ H~CO=I't ~CI. N'H CI Indomethzw.Jn

Mec~lenamale

Fig. 3. Structures of several non-steroidal anti-inflammatory agents that inhibit PGH synthase.

cyclooxygenase catalysis, replacement of Set 53° with an Ala 5~° by site-directed mutagenesis yields an enzyme with kinetic properties virtually identical to those of the native enzyme [71]. Replacement of Ser $3° with Asns3°, a group which is approximately the same size as an acetylated serine, results in the loss of cyclooxygenase but not peroxidase activity [78]. Thus, it appears that the inhibitory effect of acetylation of Ser s3° by aspirin results from placement of a bulky group at this position in the enzyme which interferes with arachidonate binding [66,71,78]. A pharmacologically important aspect of the action of aspirin is its irreversible effect. There appears to be no biochemical mechanism for hydrolyzing the acetylSet 53° ester. Thus, new PGH synthase protein synthesis is required to replace enzyme inactivated by acetylation by aspirin. In cells such as platelets which are incapable or synthesizing new PGH synthase, treatment with aspirin readers the thromboxane biosynthetic machinery nonfunctional for the lifetime of the platelet {approx. 5 days). In contrast, synthesi~ of PGH synthase protein in most other c~:lls including vascular endothelial cells is relatively rapid [i]. Thus, a net pharmacological effect of aspirin is to inhibit selectively the platelet production of TxA 2, a prothrombogenic substance, without appreciably affecting the vascular synthesis of prostacyclin, an anti-thrombogenic substance. This has been verified by pharmacokinetic studies on human volunteers [79]. Acetylation of PGH synthase is the biochemical basis for using low doses of

aspirin prophylactically and therapeutically to diminish cardiovascular pathologies that result from platelet aggregation {e.g., unstable angina) [80-85]. The mechanism(s) by which flurbiprofen, meclofenamate and indomethacin cause irreversible inhibition of cyclooxygenase activity in vitro is not well-defined. Neither indomethacin nor flurbiprofen cause covalent modification of PGH ~ynthase, but the:~e drugs do become tightly associated with the protein [73.86.87]. Moreover, following treatn at with indomethacin and then removal of all the free indomethacin, only about 5% of the initial eyclooxygenase activity is retained [73,87]. Apparently, indomethacin binds to the arachidonate substrate site reversibly with a K, of 5 p.M. Longer exposure to indomethacin then causes the enzyme to undergo a conformational change to a form that binds indomethacin even more tightly [73]. Presumably, the same inactivation process occurs with flurbiprofen and meclofenamate. Limited proteolysis experiments provide direct evidence that certain nonsteroidal anti-inflammatory agents induce conformationai changes in PGH synthase. ApoPGH synthase is readily cleaved at Arg'-77-Gly"-7~ by trypsin to produce fragments of 33 and 38 kDa [88]. Reconstitution of native enzyme by addition of heme renders the susceptible bond resistant to tryptic cleavage [89]. Certain non-steroidal anti-inflammatory agents that are cyclooxygenase inhibitors also prevent cleavage of apoPGH synthase by trypsin [731. These include indomethacin, meclofenamic acid, flurbiprofen, flufenamic acid, and ibuprofen. Of particular interest. the (S)-enantiomer of flurbiprofen is a very effective protective agent whereas the (R)-enantiomer is not. This corresponds to the relative potency of the two compounds as cyetooxygenase inhibitors and anti-inflammatory agents. The ability of nonsteroidal anti-inflammatory drugs to protect apoPGH synthase from tryptic cleavage at Arg z77 has been proposed to arise from their ability to bind to the protein in this region [73]. Whether they physically exclude trypsin or change the conformation of the protein so that the Arg277-Gly"7x bond is inaccessible is not known. However, binding of these drugs to apoprotein does not prevent binding of heine and treatment of apoprotein with indomethacin and heine simultaneously provides a higher degree of protection from tryptic cleavage than either compound alone. Thus, the anti-inflammatory compounds th'lt prevent tryptic cleavage of apoprotein do not appear to bind to the heme binding site of the protein. Salicylate is a relatively potent anti-inflammatory agent, but it is a poor eyclooxygenase inhibitor (K, > 20 mM); unlike acetylsalicylate, salicylate does not cause irreversible inactivation of the enzyme either in vitro or in vivo. The inhibitory potency of salicylate in vivo may be due to one of its metabolites, e.g., gentisic acid [65]. Recently, salicytate at very low concentrations (approx.

20 nM) has been reported to block the de nc,vo synthesis of PGH synthase in cultures of human umbilical vein endothelial cells [90]. This is a curious finding that needs to be examined in other cell types. IV. Catalytic prooerlies: ~roxida.~ reaction i V-A. Pero.xid~' substmte specificity The peroxidase activity of PGH synthase catalyzes the reduction of a variety of hydroperoxides to alcohols at the expense of a reducing cosubstrate. [he peroxidase activity of sheep or bovine vesicular gland microsomes copurifies with cyclooxygenase activity i10,11] and immunoprecipitates with monoclonal antibodies raised against ovine cydooxygenase [12]. A heme prosthetic group is essential for peroxidase activity. Apoprotein reconstituted with Fe 3+-PPIX exhibits peroxidase activity but apoprotein reconstituted with Mn 3*-pPIX exhibits less than 5% of the peroxidase activity of the native enzyme [91]. In contrast, both Fe ~ ~-PPIX and Mn~+-PP1X support cyclooxygenase activity [91]. The peroxidase of PGH synthase preferentially reduces fatty acid hydroperoxides including PGG,_ [59]. The peroxidase is much less active toward H.,O_, and almost inactive toward tertiary hydroperoxides such as cumene hydroperoxide and t-butyl-hydroperoxide [(59]. As noted earlier, similar specificity is seen with peroxide activation of cyclooxygena~,: [58]. The products of the reduction of 5-phenyl-4-pentenyl-hydroperoxide(PPHP) or 10-hydroperoxy-8,12-octadecadienoic acid by PGH synthase in the presence of reducing substrates indicate that the hydropero×ide is reduced by two electrons simultaneously [20,92]. Alcohol is produced directly without the intermediary of one-electron reduced intermediates (i.e., alkoxyt radicals). This is typical of reduction of hydroperoxides by classic heme peroxidases (e.g., horseradish peroxidase, cytochrome c peroxidase) [31. I V-B. Spectral intermediates A characteristic feature of heme peroxidases is the formation of spectroscopically detectable intermediaies during the catalytic cycle (Fig. 4). These .intermediates represent higher oxidation states of the enzyme in which the redox equivalents are contained on the heme prosthetic group 193-97]. Addition of the hydroperoxides PPHP, 15-HPETE, or PGG 2 to PGH synthase in the absence of an electron donor results in a decrease in the Soret absorbance of the resting enzyme and production of a featureless spectrum in the visible region (Fig. 5) [981. These spectral changes are complete within about 10 ms after mixing and are identical regardless of the structure of the hydroperoxide added. The decrease in

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Fig. 4, Catalytic cycle of heme-conlainingperoxidases. Intermediates I and t/ represent the spectroscopicallydetectable higher oxidation states described in the text.

intensity of the Sorer absorption is characteristic of a two-etectron oxidized heine in which the iron is in the + 4 oxidation state and the porphyrin is oxidized to a radical cation [99]. The spectrum of this species is analogous to compound I of horseradish peroxidase [95]. The formation of a two-electron oxidized heine is consistent with two-electron reduction of the hydroperoxide. Upoil standing in the presence of a peroxide ( > 100 ms), the Sorer absorbance increases in intensity and shifts from 412 nm to 420 nm (Fig. 6) [98]. Distinct ot and fl absorption maxima are evident at 527 and 557 nm. The spectrum of this intermediate is virtually superimposable on the spectrum of compound II of horseradish peroxidase and compound ES of yeast cytochrome c peroxidase [95,100,101]. The latter compounds contain a one-electron oxidized prosthetic group in which the iron is ( + 4 ) and the porphyrin is futly covalent [101]. in other words, the transformation of PGH synthase compound I to ~,ompound II is the result or one-electron reduction of the porphyrin ?adical cation of compound I. The spectrum of compound II slowly reverts to that of resting enzyme (1-2 min at 5°C) [98], Reduction of compound I! to resting enzyme is pre-

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Fig. 6. Spectrum of PGH synthase compound IL Experimentalconditions as in Fig. 5. Spectra in the Sorer region are shown at It. 22 and 132 ms after mixing, Spectra in the visible region are shown at 16, 34, and 166 ms after mixing, The arrows indicate the direction of the absorbance change with increasing time, The broken line is the native enzymespectrum. Reproduced from Ref. 98 with permission.

sumably supported by redu¢iants present in the enzyme preparation (e.g., diethyldithiocarbamate). Karthein et al. [102] reported the detection of an EPR signal at g = 2.005 concomitant with the formation of a compound 11 absorption spectrum (Fig. 7). The hyperfinc splitting pattern of the EPR signal suggests it is a tyrosyl radical as does the similarity of the spectrum to that of the tyrosyl radical of ribonucleotide reductase. Since there was no reducing substrate present in these experiments, the spectral changes were explained as the result of an intramolecular electron transfer from a tyrosyl residue on the protein to the porphyrin cation radical of compound ! (eqn. 1) [102,103]. Therefore, PGH synthas¢ compound 11 is still two

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