Identification of two cyclooxygenase active site residues, Leucine 384 and Glycine 526, that control carbon ring cyclization in prostaglandin biosynthesis

The cyclooxygenase (COX) reaction of prostaglandin (PG) biosynthesis begins with the highly specific oxygenation of arachidonic acid in the 11R configuration and ends with a 15S oxygenation to form PGG2. To obtain new insights into the mechanisms of stereocontrol of oxygenation, we mutated active site residues of human COX-2 that have potential contacts with C-11 of the reacting substrate. Although the 11R oxygenation was not perturbed, changing Leu-384 (into Phe, Trp), Trp-387 (Phe, Tyr), Phe-518 (Ile, Trp, Tyr), and Gly-526 (Ala, Ser, Thr, Val) impaired or abrogated PGG2 synthesis, and typically 11R-HETE was the main product formed. The Gly-526 and Leu-384 mutants formed, in addition, three novel products identified by LC-MS, NMR, and circular dichroism as 8,9-11,12-diepoxy-13R-(or 15R)-hydro(pero)xy derivatives of arachidonic acid. Mechanistically, we propose these arise from a free radical intermediate in which a C-8 carbon radical displaces the 9,11-endoperoxide O-O bond to yield an 8,9-11,12-diepoxide that is finally oxygenated stereospecifically in the 13R or 15R configuration. Formation of these novel products signals an arrest in the normal course of prostaglandin synthesis just prior to closing of the 5-membered carbon ring, and points to a crucial role for Leu-384 and Gly-526 in the correct positioning of the reacting fatty acid intermediate. Some of the Gly-526 and Leu-384 mutants catalyzed both formation of PGG2 (with the normal 15S configuration) and the 13R- or 15R-oxygenated diepoxides. This result suggests that oxygenation specificity can be determined by the orientation of the reacting fatty acid radical and is not a predetermined outcome based solely on the structure of the cyclooxygenase active site.     

Control of prostaglandin stereochemistry at the 15-carbon by cyclooxygenases-1 and -2. A critical role for serine 530 and valine 349.

Prostaglandin synthesis by cyclooxygenases-1 and -2 (COX-1 and COX-2) involves an initial oxygenation of arachidonic acid at C-11, followed by endoperoxide and cyclopentane ring formation, and then a second reaction with molecular oxygen in the S configuration at C-15. The resulting 15S-hydroxyl group of prostaglandins is crucial for their bioactivity. Using human COX-1 and human and murine COX-2, we have identified two amino acids located in the oxygenase active site that control the stereochemistry at C-15. The most crucial determinant is Ser-530, the residue that is acetylated by aspirin. In COX-2, site-directed mutagenesis of Ser-530 to methionine, threonine, or valine produced highly active enzymes that formed 82-95% 15R-configuration prostaglandins; these have the opposite stereochemistry at C-15 to the natural products. In COX-1, the corresponding Ser-530 mutations inactivated the enzyme. The second residue, Val-349, exerts a more subtle influence. When Val-349 was replaced by isoleucine, the mutant COX-1 and COX-2 enzymes formed 41 and 65% 15R-prostaglandins, respectively. This change was highly specific for isoleucine, as mutations of Val-349 to alanine, leucine, asparagine, or threonine did not alter or only slightly altered (< or =13%) the S-configuration at C-15. These results establish a previously unrecognized role for Ser-530 and Val-349 in maintaining the correct S stereochemistry of the carbon-15 hydroxyl group during prostaglandin synthesis. The findings may also explain the absolute conservation of Ser-530, the target of aspirin, throughout the families of cyclooxygenase enzymes.     

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

Arachidonic acid is converted to prostaglandin G(2) (PGG(2)) by the cyclooxygenase activities of prostaglandin endoperoxide H synthases (PGHSs) 1 and 2. The initial, rate-limiting step is abstraction of the 13-proS hydrogen from arachidonate which, for PGG(2) formation, is followed by insertion of O(2) at C-11, cyclization, and a second O( 2) insertion at C-15. As an accompaniment to ongoing structural studies designed to determine the orientation of arachidonate in the cyclooxygenase site, we analyzed the products formed from arachidonate by (a) solubilized, partially purified ovine (o) PGHS-1; (b) membrane-associated, recombinant oPGHS-1; and (c) a membrane-associated, recombinant active site mutant (V349L oPGHS-1) and determined kinetic values for formation of each product. Native forms of oPGHS-1 produced primarily PGG(2) but also several monohydroxy acids, which, in order of abundance, were 11R-hydroxy-5Z, 8Z,12E,14Z-eicosatetraenoic acid (11R-HETE), 15S-hydroxy-5Z,8Z,11Z, 13E-eicosatetraenoic acid (15S-HETE), and 15R-HETE. V349L oPGHS-1 formed primarily PGG(2), 15S-HETE, and 15R-HETE but only trace amounts of 11R-HETE. With native enzyme, the K(m) values for PGG(2), 11-HETE, and 15-HETE formation were each different (5.5, 12.1, and 19.4 microM, respectively); similarly, the K(m) values for PGG(2) and 15-HETE formation by V349L oPGHS-1 were different (11 and 5 microM, respectively). These results establish that arachidonate can assume at least three catalytically productive arrangements within the cyclooxygenase site of oPGHS-1 leading to PGG(2), 11R-HETE, and 15S-HETE and/or 15R-HETE, respectively. IC(50) values for inhibition of formation of the individual products by the competitive inhibitor, ibuprofen, were determined and found to be the same for a given enzyme form (i.e. 175 microM for oPGHS-1 and 15 microM for V349L oPGHS-1). These latter results are most simply rationalized by a kinetic model in which arachidonate forms various catalytically competent arrangements only after entering the cyclooxygenase active site.     

Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid

Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O(2), and two electrons to prostaglandin endoperoxide H(2) (PGH(2)). Formation of PGH(2) involves an initial oxygenation of arachidonate to yield PGG(2) catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG(2) to form PGH(2) catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co(3+)-heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG(2) versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually make less but measurable contributions to optimal catalytic efficiency.     

Non-redundant functions of cyclooxygenases: oxygenation of endocannabinoids

The two cyclooxygenase (COX) enzymes catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides, which are the common intermediates in the biosynthesis of the bioactive lipids prostaglandins and thromboxane. COX-1 and COX-2 are approximately 60% identical in amino acid sequence, exhibit highly homologous three-dimensional structures, and appear functionally similar at the biochemical level. Recent work has uncovered a subtle functional difference between the two enzymes, namely the ability of COX-2 to efficiently utilize neutral derivatives (esters and amides) of arachidonic acid as substrates. Foremost among these neutral substrates are the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamide. This raises the possibility that COX-2 oxygenation plays a role in a novel signaling pathway dependent on agonist-induced release of endocannabinoids and their selective oxygenation by COX-2. Among the products of COX-2 oxygenation of endocannabinoids are glyceryl prostaglandins, some of which (e.g. glyceryl prostaglandin E(2) and glyceryl prostaglandin I(2)) exhibit interesting biological activities in inflammatory, neurological, and vascular systems. These compounds are produced in intact cells stimulated with physiological agonists and have been isolated from in vivo sources. Important concepts relevant to the hypothesis of a COX-2-selective signaling pathway are presented.     

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid 2-arachidonylglycerol

The endocannabinoid, 2-arachidonylglycerol (2-AG), is an endogenous ligand for the central (CB1) and peripheral (CB2) cannabinoid receptors and has been shown to be efficiently and selectively oxygenated by cyclooxygenase (COX)-2. We have investigated 2-AG/COX-2 interactions through site-directed mutagenesis. An evaluation of more than 20 site-directed mutants of murine COX-2 has allowed for the development of a model of 2-AG binding within the COX-2 active site. Most strikingly, these studies have identified Arg-513 as a critical determinant in the ability of COX-2 to efficiently generate prostaglandin H(2) glycerol ester, explaining, in part, the observed isoform selectivity for this substrate. Mutational analysis of Leu-531, an amino acid located directly across from Arg-513 in the COX-2 active site, suggests that 2-AG is shifted in the active site away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Despite this difference, aspirin-treated COX-2 oxygenates 2-AG to afford 15-hydroxyeicosatetraenoic acid glycerol ester in a reaction analogous to the C-15 oxygenation of arachidonic acid observed with acetylated COX-2. Finally, the differences in substrate binding do not alter the stereospecificity of the cyclooxygenase reaction; 2-AG-derived and arachidonic acid-derived products share identical stereochemistry.     

On the relationships of substrate orientation, hydrogen abstraction, and product stereochemistry in single and double dioxygenations by soybean lipoxygenase-1 and its Ala542Gly mutant

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-(3)H- and 11S-(3)H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S, 15S- and 8S, 15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R, 15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration "partner" of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.     

Catalytic convergence of manganese and iron lipoxygenases by replacement of a single amino Acid

Lipoxygenases (LOXs) contain a hydrophobic substrate channel with the conserved Gly/Ala determinant of regio- and stereospecificity and a conserved Leu residue near the catalytic non-heme iron. Our goal was to study the importance of this region (Gly(332), Leu(336), and Phe(337)) of a lipoxygenase with catalytic manganese (13R-MnLOX). Recombinant 13R-MnLOX oxidizes 18:2n-6 and 18:3n-3 to 13R-, 11(S or R)-, and 9S-hydroperoxy metabolites (～80-85, 15-20, and 2-3%, respectively) by suprafacial hydrogen abstraction and oxygenation. Replacement of Phe(337) with Ile changed the stereochemistry of the 13-hydroperoxy metabolites of 18:2n-6 and 18:3n-3 (from ～100% R to 69-74% S) with little effect on regiospecificity. The abstraction of the pro-S hydrogen of 18:2n-6 was retained, suggesting antarafacial hydrogen abstraction and oxygenation. Replacement of Leu(336) with smaller hydrophobic residues (Val, Ala, and Gly) shifted the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3. Replacement of Gly(332) and Leu(336) with larger hydrophobic residues (G332A and L336F) selectively augmented dehydration of 13R-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid and increased the oxidation at C-13 of 18:1n-6. We conclude that hydrophobic replacements of Leu(336) can modify the hydroperoxide configurations at C-9 with little effect on the R configuration at C-13 of the 18:2n-6 and 18:3n-3 metabolites. Replacement of Phe(337) with Ile changed the stereospecific oxidation of 18:2n-6 and 18:3n-3 with formation of 13S-hydroperoxides by hydrogen abstraction and oxygenation in analogy with soybean LOX-1.     

Detection and cellular localization of 12R-lipoxygenase in human tonsils

Formation of the 12R-lipoxygenase product, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE), has been detected previously only in human skin (Boeglin et al. (1998) Proc. Natl. Acad. Sci. USA 95, 6744). The unexpected appearance of an EST sequence (AA649213) for human 12R-lipoxygenase from germinal center B lymphocytes purified from human tonsils prompted our search for the existence of the enzyme in this novel source. Incubation of [1-14C]arachidonic acid with homogenates of human tonsillar tissue yielded mixtures of radiolabeled 12-HETE and 15-HETE. Stereochemical analysis showed varying ratios of 12S- and 12R-HETE, while 15-HETE was exclusively of the S-configuration. Using stereospecifically labeled [10S-3H]- and [10R-3H]arachidonic acid substrates we detected pro-R hydrogen abstraction at carbon 10 associated with formation of 12R-HETE. This mechanistic evidence implicates a 12R-lipoxygenase in the biosynthesis of 12R-HETE. The mRNA for the enzyme was identified in tonsils by RT-PCR and Northern analysis. The cellular distribution was established by in situ hybridization. Unexpectedly, hybridization was not observed in the lymphocytes of the germinal centers. Specific reaction was restricted to squamous epithelial cells, including the epithelium lining the tonsillar crypts. In this location the 12R-lipoxygenase might help regulate differentiation of the epithelium or participate in lymphocyte- epithelial cell interactions.     

Mechanism of prostaglandin biosynthesis in rabbit kidney medulla. A rate-limiting step and the differential stimulatory actions of L-adrenaline and glutathione.

Microsomal prostaglandin synthase (EC 1.14.99.1) from rabbit kidney medulla was assayed with [5,6,8,9,11,12,14,15-3H]-and [1-14C]-arachidonic acid as the substrate. The ratios of prostaglandin F2 alpha to prostaglandin E2 and to prostaglandin D2 were determined by both 3H and 14C labelling. When 3H was used as a label the ratios were much higher than with 14C labelling indicating that the removal of hydrogen at C-9 or C-11 was the rate-limiting step in the biosynthesis of prostaglandin E2 or prostaglandin D2. This finding shows that the octatritiated arachidonic acid is not the appropriate substrate marker for studying the regulation of the synthesis of different prostaglandins by various agents. When the enzyme assay was carried out in the presence of SnCL2, which was capable of accumulating exclusively prostaglandin F2alpha at the expenses of prostaglandin E2 and prostaglandin D2, the addition of L-adrenaline to the microsomal fraction either alone or with reduced glutathione equally stimulated the formation of prostaglandin F2alpha, whereas the addition of reduced glutathione to the microsomal fraction either alone or with L-adrenaline produced no additional effect. These results suggest that endoperoxide is formed as the common intermediate for the biosynthesis of three different prostaglandins in rabbit kidney medulla, and that L-adrenaline stimulates the synthesis of endoperoxide, whereas reduced glutathione facilitates the formation of prostaglandins from endoperoxide.     

Synthesis and applications of stereospecifically (3)H-labeled arachidonic acids as mechanistic probes for lipoxygenase and cyclooxygenase catalysis

Stereospecifically (3)H-labeled substrates are useful tools in studying the mechanism of hydrogen abstractions involved in the oxygenation of polyunsaturated fatty acids. Here, we describe modified methods for the synthesis of arachidonic acids labeled with a single chiral tritium on the methylene groups at carbons 10 or 13. The appropriate starting material is a ketooctadecanoic acid which is prepared from an unsaturated C18 fatty acid precursor or by total synthesis. The (3)H label is introduced by NaB(3)H(4) reduction and the resulting tritiated hydroxy fatty acid then is tosylated, separated into the enantiomers by chiral phase HPLC, and subsequently transformed into stearic acids. A variety of stereospecifically labeled unsaturated fatty acids are obtained using literature methods of microbial transformation with the fungus Saprolegnia parasitica. Two applications are described: (i) In incubations of [10S-(3)H]- and [10R-(3)H]arachidonic acids in human psoriatic scales we show that a 12R-lipoxygenase accounts not only for synthesis of the major product 12R-HETE, but it contributes also, through subsequent isomerization, to the minor amounts of 12S-HETE. (ii) The [10R-(3)H]- and [10S-(3)H]arachidonic acids were also used to demonstrate that prostaglandin ring formation by cyclooxygenases does not involve carbocation formation at C-10 of arachidonic acid as was hypothesized recently.     

Metabolism of linoleic and arachidonic acids in VX2 carcinoma tissue: Identification of monohydroxy octadecadienoic acids and monohydroxy eicosatetraenoic acids

The metabolism of arachidonic and linoleic acids by VX₂ carcinoma tissue was determined. Prostaglandin E₂ was the major metabolic product of arachidonic acid in the neoplastic tissue. Minor products accounting for 3– 8% of arachidonic acid metabolism were 11-hydroxy-5, 8, 12, 14-eicosatetraenoic acid (11-HETE) and 15-hydroxy-5, 8, 11, 13-eicosatetraenoic acid (15-HETE). Linoleic acid was converted to a mixture of 9-hydroxy-10, 12-octadecadienoic acid (9-HODD) and 13-hydroxy-9, 11-octadecadienoic acid (13-HODD). The conversion of linoleic acid to monohydroxy C-18 fatty acids varied from 40–80% 9-HODD and 20–60% 13-HODD in tumor tissue harvested from different animals. The quantity of monohydroxy C-18 fatty acids biosynthesized by VX₂ carcinoma tissue from endogenous linoleic acid equals or exceeds that of prostaglandin E₂ biosynthesis from endogenous arachidonic acid. The presence of a hydroxyl group adjacent to a conjugated diene suggest that the monohydroxy C-18 and monohydroxy C-20 fatty acids were formed via the action of lipoxygenase-like enzymes. These lipoxygenase-like reactions are inhibited by indomethacin in a concentration-dependent fashion similar to the inhibition of prostaglandin E₂ biosynthesis. The enzymes catalyzing the lipoxygenase-like reactions of linoleic and arachidonic acids are localized in the microsomal fraction of VX₂ carcinoma tissue. These data suggest that the lipoxygenase-like reactions are catalyzed by fatty acid cyclooxygenase and that there are two major pathways of fatty acid cyclooxygenase metabolism of polyenoic fatty acids in the neoplastic tissue. One pathway involves the formation of prostaglandin E₂ via cyclic endoperoxy intermediates. The second pathway involves the formation of monohydroxy C-18 fatty acids from linoleic acid via lipoxygenase-like reactions.     

Lipoxygenation activity of purified prostaglandin-forming cyclooxygenase.

Purified cyclooxygenase, a single enzyme which catalyzes the formation of endoperoxide from arachidonic acid (20:4) in a bis(dioxygenase) reaction, is capable of oxygenating eicosadienoic acid (20:2) at C-11 in a single dioxygenase reaction. The partial oxygenation of 20:2 resembles the formation of prostaglandin from 20:4, with both oxygenation reactions exhibiting similar pH optima, substrate Km values, and cofactor effects including a need for peroxide and an absolute requirement for heme. In addition, those processes known to destroy 20:4 oxygenase activity, such as heat inactivation, inactivation with anti-inflammatory drugs, and turnover-mediated inactivation, have equally destructive effects on 20:2 oxygenase activity. Thus, both oxygenations are catalyzed by one enzyme. All of the above similarities for 20:2 and 20:4 oxygenation demonstrate that C-11 oxygenation is an integral rate-limiting step of cyclooxygenase action rather than a separate reaction resembling that of plant lipoxygenase.     

Mutational and X-ray crystallographic analysis of the interaction of dihomo-gamma -linolenic acid with prostaglandin endoperoxide H synthases

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) catalyze the committed step in prostaglandin biosynthesis. Both isozymes can oxygenate a variety of related polyunsaturated fatty acids. We report here the x-ray crystal structure of dihomo-gamma-linolenic acid (DHLA) in the cyclooxygenase site of PGHS-1 and the effects of active site substitutions on the oxygenation of DHLA, and we compare these results to those obtained previously with arachidonic acid (AA). DHLA is bound within the cyclooxygenase site in the same overall L-shaped conformation as AA. C-1 and C-11 through C-20 are in the same positions for both substrates, but the positions of C-2 through C-10 differ by up to 1.74 A. In general, substitutions of active site residues caused parallel changes in the oxygenation of both AA and DHLA. Two significant exceptions were Val-349 and Ser-530. A V349A substitution caused an 800-fold decrease in the V(max)/K(m) for DHLA but less than a 2-fold change with AA; kinetic evidence indicates that C-13 of DHLA is improperly positioned with respect to Tyr-385 in the V349A mutant thereby preventing efficient hydrogen abstraction. Val-349 contacts C-5 of DHLA and appears to serve as a structural bumper positioning the carboxyl half of DHLA, which, in turn, positions properly the omega-half of this substrate. A V349A substitution in PGHS-2 has similar, minor effects on the rates of oxygenation of AA and DHLA. Thus, Val-349 is a major determinant of substrate specificity for PGHS-1 but not for PGHS-2. Ser-530 also influences the substrate specificity of PGHS-1; an S530T substitution causes 40- and 750-fold decreases in oxygenation efficiencies for AA and DHLA, respectively.     

Stereospecific elimination of hydrogen at C-10 in eicosapentaenoic acid during the conversion to leukotriene C5

(10L)- and (10D)-[1-14C, 10-3H]5,8,11,14,17-eicosapentaenoic acids were synthesized to investigate mechanistic and stereochemical aspects of leukotriene biosynthesis. Experiments with mastocytoma cells showed that a hydrogen is stereospecifically eliminated from C-10 during the conversion of eicosapentaenoic acid to leukotriene C5. The hydrogen lost has the pro-S (D) configuration. 5-Hydroxy-6,8,11,14,17-eicosapentaenoic acid, formed in the same experiments, was enriched in tritium when the (10D), but not when the (10L), isomer of labeled eicosapentaenoic acid was used. This indicates that oxygenation of the acid at C-5 occurred before the elimination of hydrogen and suggests that removal of the pro-S hydrogen at C-10 in 5-hydroperoxy-6,8,11,14,17-eicosapentaenoic acid initiates its transformation to trans-5(S),6(S)-oxido-7,9-trans-11,14,17-cis-eicosapentaenoic acid (leukotriene A5).     

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide

The endocannabinoid arachidonylethanolamide (AEA, anandamide) is an endogenous ligand for the cannabinoid receptors and has been shown to be oxygenated by cyclooxygenase-2 (COX-2). We examined the structural requirements for COX-mediated, AEA oxygenation using a number of substrate analogues and site-directed mutants of COX-2. Fourteen AEA analogues were synthesized and tested as COX substrates. These studies identified the hydroxyl moiety of AEA as a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation. In addition, these studies suggest that subtle structural modifications of AEA analogues near the ethanolamide moiety can result in pronounced changes in their ability to serve as COX-2 substrates. Site-directed mutagenesis studies have permitted the development of a model of AEA binding within the COX-2 active site. As with arachidonic acid, the omega-terminus of AEA binds in a hydrophobic alcove near the top of the COX-2 active site. The polar ethanolamide moiety of AEA, like the carboxylate of arachidonate, interacts with Arg-120 at the bottom of the COX-2 active site. Mutation of Tyr-385 prevents AEA oxygenation, suggesting that, as in the case of other COX substrates, AEA metabolism is initiated by Tyr-385-mediated hydrogen abstraction. Thus, AEA binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. However, important differences have been identified that account for the isoform selectivity of AEA oxygenation. Importantly, the COX-2 side pocket and Arg-513 in particular are critical determinants of the ability of COX-2 to efficiently generate prostaglandin H(2) ethanolamide. The reduced efficiency of COX-1-mediated, AEA oxygenation can thus be explained by the absence of an arginine residue at position 513 in this isoform. Mutational analysis of Leu-531, an amino acid located directly across from the COX-2 side pocket, suggests that AEA is shifted away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Coupled with earlier observations with the endocannabinoid 2-arachidonylglycerol, these results indicate that one possible function of the highly conserved COX-2 active site side pocket is to promote endocannabinoid oxygenation.     

13-Methylarachidonic Acid Is a Positive Allosteric Modulator of Endocannabinoid Oxygenation by Cyclooxygenase

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing k_cat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.     

Leukotriene formation by a purified reticulocyte lipoxygenase enzyme. Conversion of arachidonic acid and 15-hydroperoxyeicosatetraenoic acid to 14, 15-leukotriene A4

The purified lipoxygenase of rabbit reticulocytes converts arachidonic acid at 0 degrees C to 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and to 12-hydroperoxyeicosatetraenoic acid (12-HPETE) via reactions which involve hydrogen abstraction at C-13 and C-10, respectively. At 37 degrees C the enzyme converts arachidonic acid to additional products which were identified as 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid, 8,15-dihydroperoxy-5,9,11,13- and 5,15-dihydroperoxy-6, 6,8,11,13-eicosatetraenoic acids (8,15-diHPETE and 5,15-HPETE, respectively) and diastereoisomers of 8,15-dihydroxy-5,9,11,13-eicosatetraenoic acid (8,15-diHPETEs). The 8,15- and 5,15-diHPETEs were formed by double lipoxygenation since each incorporated 2 molecules of 18O2 and since their synthesis from 15-HPETE was blocked under anaerobic conditions. The 8,15-diHETEs each incorporated 18O from 18O2 at C-15 and were found to arise from nonenzymatic hydrolysis of an epoxytriene which was identified as 14,15-leukotriene A4 by trapping in acidic methanol. This compound was a major product of 15-HPETE in anaerobic incubations. The conversion of 15-HPETE to 14,15-leukotriene A4 was inhibited by the lipoxygenase inhibitors nordihydroguairetic acid and 5,8,11,14-eicosatetraynoic acid. The 14,15-leukotriene A4 synthase and 15-lipoxygenase activities were inhibited by 5,8,11,14-eicosatetraynoic acid in a similar time-dependent manner. The results support a mechanism whereby 14,15-leukotriene A4 is synthesized from 15-HPETE by a further enzymatic step carried out by the reticulocyte 15-lipoxygenase via hydrogen abstraction at C-10 and a redox cycle of the non-heme iron atom of the enzyme.     

Properties of prostaglandin synthetase of rabbit kidney medulla.

The formation in vitro of prostaglandins E2, D2, and F2alpha from arachidonic acid by rabbit kidney medulla homogenate or microsomal fraction is markedly affected by the composition of the incubation medium employed. Optimal biosynthesis is obtained in 0.1 M potassium phosphate buffer, with the optimum pH being 8.0--8.8. Under these conditions prostaglandin formation is linear up to arachidonic acid concentration of 30 muM. The initial rate of formation of prostaglandin E2 + prostaglandin D2 is 3--4 times higher than that of prostaglandin F2alpha. Reduced glutathione (1 mM) did not affect the biosynthesis by medulla homogenate and produced only small stimulation of the biosynthesis by microsomal powder. Hydroquinone produced a small stimulation at a low concentration of 0.005 mM, and a strong inhibition at concentrations of 0.1 mM or higher. Addition of bovine serum albumin (0.1%) reduced the microsomal biosynthesis of prostaglandins by approximately 80%. Addition of boiled homogenate or boiled 140 000 X g supernatant produced small stimulation of microsomal biosynthesis while 140 000 X g supernatant (not boiled) caused small inhibition which was not dose-related. It appears that rabbit kidney prostaglandin-synthetase converts arachidonic acid to prostaglandins E2 and F2alpha in comparable amounts, without apparent need for a cytoplasmic soluble cofactor or specific reducing agents.     

On singular or dual positional specificity of lipoxygenases. The number of chiral products varies with alignment of methylene groups at the active site of the enzyme

We tested a simple model which explains the singular or dual specificity of lipoxygenases. The dual specificity considered here is typified by the oxygenation of arachidonic acid by the reticulocyte lipoxygenase: two chiral products are formed (12S- and 15S-hydroperoxides, ratio approximately 1:9) via hydrogen abstraction from two separate methylene groups (C-10 and C-13). The rate-limiting step is known to involve this hydrogen abstraction, and we assumed that alignment of the methylenes with the hydrogen acceptor on the enzyme is critical in terms of reaction rate and positional specificity. Optimal alignment will be associated with a fast rate of reaction and formation of a single chiral product. A shift in position of the double bonds (and hence of the methylene groups) should be associated with a slower rate of reaction and formation of two chiral products; two methylenes are now able to react, although neither has perfect alignment. We tested this idea using two lipoxygenases and polyenoic fatty acids differing in the number and position of the double bonds. Optimal substrates for the soybean lipoxygenase had a doubly allylic methylene in the n-8 position, while the reticulocyte enzyme preferred substrates with a n-9 methylene. These substrates were converted to a single chiral product. With both enzymes, the other series of substrates reacted more slowly and were converted to two chiral products. We conclude that alignment of methylene groups of the substrate at the active site is a major determinant of the reaction rate and the singular or dual specificity of lipoxygenases.