Polymorphic human prostaglandin H synthase-2 proteins and their interactions with cyclooxygenase substrates and inhibitors.

The cyclooxygenase (COX) activity of prostaglandin H synthase-2 (PGHS-2) is implicated in colorectal cancer and is targeted by nonsteroidal anti-inflammatory drugs (NSAIDs) and dietary n-3 fatty acids. We used purified, recombinant proteins to evaluate the functional impacts of the R228H, E488G, V511A and G587R PGHS-2 polymorphisms on COX activity, fatty acid selectivity and NSAID actions. Compared to wild-type PGHS-2, COX activity with arachidonate was ∼20% lower in 488G and ∼20% higher in 511A. All variants showed time-dependent inhibition by the COX-2-specific inhibitor (coxib) nimesulide, but 488G and 511A had 30-60% higher residual COX activity; 511A also showed up to 70% higher residual activity with other time-dependent inhibitors. In addition, 488G and 511A differed significantly from wild type in Vmax values with the two fatty acids: 488G showed ∼20% less and 511A showed ∼20% more discrimination against eicosapentaenoic acid. The Vmax value for eicosapentaenoate was not affected in 228H or 587R, nor were the Km values or the COX activation efficiency (with arachidonate) significantly altered in any variant. Thus, the E488G and V511A PGHS-2 polymorphisms may predict who will most likely benefit from interventions with some NSAIDs or n-3 fatty acids.

Arabidopsis thaliana fatty acid alpha-dioxygenase-1: evaluation of substrates, inhibitors and amino-terminal function.

Plant alpha dioxygenases (PADOX) convert fatty acids to 2-hydroperoxy products that are important in plant signaling pathways. The PADOX amino-terminal domain is distinct from that in other myeloperoxidase-family hemoproteins, and the positional specificity and prosthetic group of PADOX distinguish them from the non-heme iron plant lipoxygenases. The constraints of the PADOX active site on potential substrates are poorly understood and only limited structure-function and mechanistic information is available for these enzymes. We developed several bacterial and insect cell systems for expression of recombinant Arabidopsis thaliana PADOX1 and evaluated the enzyme's substrate and inhibitor profiles and explored the functional role of the amino-terminal domain. Substrate specificity studies gave the following relative oxygenase activity values: linolenate, 1.00; linoleate, 0.95; oleate, 0.84; palmitoleate, 0.69; myristate, 0.23; palmitate, 0.17; and gamma-linolenate, 0.16. Methyl esters of myristate, linoleate and linolenate were not oxygenated. 3-Thiamyristate was the only oxygenase substrate that produced pronounced enzyme self-inactivation during catalysis. 3,4-Dehydromyristate inactivated the oxygenase without appreciable oxygen consumption. Several compounds inhibited oxygenase activity, including catechol (K(i) approximately 90 microM), divalent zinc ion (K(i) approximately 50 microM), N,N,N',N'-tetramethyl-p-phenylenediamine (K(i) approximately 20 microM) and cyanide ion (K(i) approximately 5 microM). Zinc ion did not change the K(m) values for linoleate or oxygen, or the K(i) value for cyanide, indicating that zinc acts at a distinct site from the other compounds. Gel-filtration chromatography revealed considerable variation in oligomeric state of recombinant PADOX1 produced in the various expression systems, but oligomeric state was not correlated with activity. Deletion of the first eight or fourteen PADOX1 residues in a NuSA-PADOX1 fusion protein led to 13 and 83% decreases in activity, respectively, indicating the N-terminal region is important for normal catalytic activity.

Peroxidase self-inactivation in prostaglandin H synthase-1 pretreated with cyclooxygenase inhibitors or substituted with mangano protoporphyrin IX.

Self-inactivation imposes an upper limit on bioactive prostanoid synthesis by prostaglandin H synthase (PGHS). Inactivation of PGHS peroxidase activity has been found to begin with Intermediate II, which contains a tyrosyl radical. The structure of this radical is altered by cyclooxygenase inhibitors, such as indomethacin and flurbiprofen, and by replacement of heme by manganese protoporphyrin IX (forming MnPGHS-1). Peroxidase self-inactivation in inhibitor-treated PGHS-1 and MnPGHS-1 was characterized by stopped-flow spectroscopic techniques and by chromatographic and mass spectrometric analysis of the metalloporphyrin. The rate of peroxidase inactivation was about 0.3 s(-)1 in inhibitor-treated PGHS-1 and much slower in MnPGHS-1 (0.05 s(-)1); as with PGHS-1 itself, the peroxidase inactivation rates were independent of peroxide concentration and structure, consistent with an inactivation process beginning with Intermediate II. The changes in metalloporphyrin absorbance spectra during inactivation of inhibitor-treated PGHS-1 were similar to those observed with PGHS-1 but were rather distinct in MnPGHS-1; the kinetics of the spectral transition from Intermediate II to the next species were comparable to the inactivation kinetics in each case. In contrast to the situation with PGHS-1 itself, significant amounts of heme degradation occurred during inactivation of inhibitor-treated PGHS-1, producing iron chlorin and heme-protein adduct species. Structural perturbations at the peroxidase site (MnPGHS-1) or at the cyclooxygenase site (inhibitor-treated PGHS-1) thus can influence markedly the kinetics and the chemistry of PGHS-1 peroxidase inactivation.

Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase.

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) catalyze the first two steps in the biosynthesis of prostaglandins. Resonance Raman spectroscopy was used to characterize the PGHS heme active site and its immediate environment. Ferric PGHS-1 has a predominant six-coordinate high-spin heme at room temperature, with water as the sixth ligand. The proximal histidine ligand (or the distal water ligand) of this hexacoordinate high-spin heme species was reversibly photolabile, leading to a pentacoordinate high-spin ferric heme iron. Ferrous PGHS-1 has a single species of five-coordinate high-spin heme, as evident from nu(2) at 1558 cm(-1) and nu(3) at 1471 cm(-1). nu(4) at 1359 cm(-1) indicates that histidine is the proximal ligand. A weak band at 226-228 cm(-1) was tentatively assigned as the Fe-His stretching vibration. Cyanoferric PGHS-1 exhibited a nu(Fe)(-)(CN) line at 446 cm(-1) and delta(Fe)(-)(C)(-)(N) at 410 cm(-1), indicating a "linear" Fe-C-N binding conformation with the proximal histidine. This linkage agrees well with the open distal heme pocket in PGHS-1. The ferrous PGHS-1 CO complex exhibited three important marker lines: nu(Fe)(-)(CO) (531 cm(-1)), delta(Fe)(-)(C)(-)(O) (567 cm(-1)), and nu(C)(-)(O) (1954 cm(-1)). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the nu(Fe)(-)(CO)/nu(C)(-)(O) correlation curve for heme proteins and porphyrins with a proximal histidine or imidazolate, suggesting an extremely weak bond between the heme iron and the proximal histidine in PGHS-1. At alkaline pH, PGHS-1 is converted to a second CO binding conformation (nu(Fe)(-)(CO): 496 cm(-1)) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.

Prostaglandin H synthase. Effects of peroxidase cosubstrates on cyclooxygenase velocity.

Many cosubstrates for the peroxidase activity of prostaglandin H synthase-1 (PGHS-1) have been reported to produce a large (2-7-fold) increase in the cyclooxygenase velocity in addition to a substantial increase in the number of cyclooxygenase catalytic turnovers. The large stimulation of cyclooxygenase velocity has become an important criterion for evaluation of putative PGHS reaction mechanisms. This criterion has been a major weakness of branched-chain tyrosyl radical mechanisms, which correctly predict many other cyclooxygenase characteristics. Our computer simulations based on a branched-chain mechanism indicated that the uncorrected oxygen electrode signals commonly used to monitor activity can seriously overestimate the effects of cosubstrate on cyclooxygenase velocity. The simulation results prompted re-examination of the effect of several cosubstrates (phenol, acetaminophen, N,N,N',N'-tetramethylphenylenediamine, and Trolox) on PGHS-1 cyclooxygenase velocity. Cyclooxygenase kinetics were examined at reduced temperature or elevated pH, where the oxygen electrode signal can be corrected to provide reliable oxygen consumption trajectories. The cosubstrates produced only a slight (10-60%) stimulation of the cyclooxygenase velocity. Peroxidase cosubstrates thus have a much smaller stimulatory effect on cyclooxygenase velocity than previously reported. This corrects a longstanding misperception of cosubstrate effects, provides more realistic kinetic constraints on PGHS mechanisms, and removes what was a major deficiency of branched-chain tyrosyl radical mechanisms.

Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1.

The tyrosyl radicals generated in reactions of ethyl hydrogen peroxide with both native and indomethacin-pretreated prostaglandin H synthase 1 (PGHS-1) were examined by low-temperature electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. In the reaction of peroxide with the native enzyme at 0 degrees C, the tyrosyl radical EPR signal underwent a continuous reduction in line width and lost intensity as the incubation time increased, changing from an initial, 35-G wide doublet to a wide singlet of slightly smaller line width and finally to a 25-G narrow singlet. The 25-G narrow singlet produced by self-inactivation was distinctly broader than the 22-G narrow singlet obtained by indomethacin treatment. Analysis of the narrow singlet EPR spectra of self-inactivated and indomethacin-pretreated enzymes suggests that they reflect conformationally distinct tyrosyl radicals. ENDOR spectroscopy allowed more detailed characterization by providing hyperfine couplings for ring and methylene protons. These results establish that the wide doublet and the 22-G narrow singlet EPR signals arise from tyrosyl radicals with different side-chain conformations. The wide-singlet ENDOR spectrum, however, is best accounted for as a mixture of native wide-doublet and self-inactivated 25-G narrow-singlet species, consistent with an earlier EPR study [DeGray et al. (1992) J. Biol. Chem. 267, 23583-23588]. We conclude that a tyrosyl residue other than the catalytically essential Y385 species is most likely responsible for the indomethacin-inhibited, narrow-singlet spectrum. Thus, this inhibitor may function by redirecting radical formation to a catalytically inactive side chain. Either radical migration or conformational relaxation at Y385 produces the 25-G narrow singlet during self-inactivation. Our ENDOR data also indicate that the catalytically active, wide-doublet species is not hydrogen bonded, which may enhance its reactivity toward the fatty-acid substrate bound nearby.

Distinct influences of carboxyl terminal segment structure on function in the two isoforms of prostaglandin H synthase.

The cyclooxygenase activity of the two prostaglandin H synthase (PGHS) isoforms, PGHS-1 and -2, is a major control element in prostanoid biosynthesis. The two PGHS isoforms have 60% amino acid identity, with prominent differences near the C-terminus, where PGHS-2 has an additional 18-residue insert. Some mutations of the C-terminal residue in PGHS-1 and -2 have been found to disrupt catalytic activity and/or intracellular targeting of the proteins, but the relationship between C-terminal structure and function in the two isoforms has been poorly defined. Crystallographic data indicate the PGHS-1 and -2 C-termini are positioned to interact with the endoplasmic reticulum (ER) membrane, although the C-terminal segment structure was not resolved for either isoform. We constructed a series of C-terminal substitution, deletion, and insertion mutants of human PGHS-1 and -2 and evaluated the effects on cyclooxygenase activity and intracellular targeting in transfected COS-1 cells expressing the recombinant proteins. PGHS-1 cyclooxygenase activity was strongly disrupted by C-terminal substitutions and deletions, but not by elongation of the C-terminal segment, even when the ultimate residue was altered. Similar alterations to PGHS-2 had markedly less effect on cyclooxygenase activity. The results indicate that the functioning of the longer C-terminal segment in PGHS-2 is distinctly more tolerant of structural change than the shorter PGHS-1 C-terminal segment. C-Terminal substitutions or deletions did not change the subcellular localization of either isoform, even at short times after transfection, indicating that neither C-terminal segment contains indispensable intracellular targeting signals.

Hydroperoxide dependence and cooperative cyclooxygenase kinetics in prostaglandin H synthase-1 and -2.

Prostaglandin H synthase isoform-1 (PGHS-1) cyclooxygenase activity has a cooperative response to arachidonate concentration, whereas the second isoform, PGHS-2, exhibits saturable kinetics. The basis for the cooperative PGHS-1 behavior and for the difference in cooperativity between the isoforms was unclear. The two cyclooxygenase activities have different efficiencies of feedback activation by hydroperoxide. To determine whether the cooperative kinetics were governed by the feedback activation characteristics, we examined the cyclooxygenase activities under conditions where feedback activation was either assisted (by exogenous peroxide) or impaired (by replacement of heme with mangano protoporphyrin IX to form MnPGHS-1 and -2). Heme replacement increased PGHS-1 cyclooxygenase cooperativity and changed PGHS-2 cyclooxygenase kinetics from saturable to cooperative. Peroxide addition decreased or abolished cyclooxygenase cooperativity in PGHS-1, MnPGHS-1, and MnPGHS-2. Kinetic simulations predicted that cyclooxygenase cooperativity depends on the hydroperoxide activator requirement and initial peroxide concentration, consistent with observed behavior. The results indicate that PGHS-1 cyclooxygenase cooperativity originates in the feedback activation kinetics and that the cooperativity difference between the isoforms can be explained by the difference in feedback activation loop efficiency. This linkage between activation efficiency and cyclooxygenase cooperativity indicates an interdependence between fatty acid and hydroperoxide levels in controlling the synthesis of potent prostanoid mediators.

Rapid kinetics of tyrosyl radical formation and heme redox state changes in prostaglandin H synthase-1 and -2.

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2. Rapid-freeze EPR and stopped-flow were used to characterize tyrosyl radical kinetics in PGHS-1 and -2 reacted with ethyl hydrogen peroxide. In PGHS-1, a wide doublet tyrosyl radical (34-35 G) was formed by 4 ms, followed by transition to a wide singlet (33-34 G); changes in total radical intensity paralleled those of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but transition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS-1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the Intermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesulide-treated PGHS-2 rapidly formed narrow singlet EPR (25-26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the indomethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical formed in concert with Intermediate II, but later persisted while Intermediate II relaxed. These results substantiate the kinetic competence of a tyrosyl radical as the catalytic intermediate for both PGHS isoforms and also indicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.

Comparison of the peroxidase reaction kinetics of prostaglandin H synthase-1 and -2.

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and -2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency. The kinetics of formation of Intermediate I (an Fe(IV) species with a porphyrin free radical) and Intermediate II (an Fe(IV) species with a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygenase catalysis) were monitored at 4 degrees c by stopped flow spectrophotometry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetraenoic acid, the rate constant for Intermediate I formation (k1) was 2.3 x 10(7) M-1 s-1 for PGHS-1 and 2.5 x 10(7) M-1 s-1 for PGHS-2, indicating that the isoforms have similar initial reactivity with this lipid hydroperoxide. For PGHS-1, the rate of conversion of Intermediate I to Intermediate II (k2) became the limiting factor when the hydroperoxide level was increased, indicating a rate constant of 10(2)-10(3) s-1 for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition between Intermediates I and II was not rate-limiting even at the highest hydroperoxide concentrations tested, indicating that the k2 value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that faster formation of the active cyclooxygenase species (Intermediate II) or increased stability of the active species increases the resistance of the cyclooxygenase to inhibition by the intracellular hydroperoxide scavenger, glutathione peroxidase. Kinetic differences between the PGHS isoforms in forming or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.

A mechanistic study of self-inactivation of the peroxidase activity in prostaglandin H synthase-1.

Prostaglandin H synthase (PGHS) is a self-activating and self-inactivating enzyme. Both the peroxidase and cyclooxygenase activities have a limited number of catalytic turnovers. Sequential stopped-flow measurements were used to analyze the kinetics of PGHS-1 peroxidase self-inactivation during reaction with several different hydroperoxides. The inactivation followed single exponential kinetics, with a first-order rate constant of 0.2-0.5 s-1 at 24 degrees C. This rate was independent of the peroxide species and concentration used, strongly suggesting that the self-inactivation process originates after formation of Compound I and probably with Intermediate II, which contains an oxyferryl heme and a tyrosyl radical. Kinetic scan and rapid scan experiments were used to monitor the heme changes during the inactivation process. The results from both experiments converged to a simple, linear, two-step mechanism in which Intermediate II is first converted in a faster step (0.5-2 s-1) to a new compound, Intermediate III, which undergoes a subsequent slower (0.01-0.05 s-1) transition to a terminal species. Rapid-quench and high pressure liquid chromatography analysis indicated that Intermediate III likely retains an intact heme group that is not covalently linked with the PGHS-1 protein.

Expression, purification, and spectroscopic characterization of human thromboxane synthase.

Thromboxane A2 (TXA2) is a potent inducer of vasoconstriction and platelet aggregation. Large scale expression of TXA2 synthase (TXAS) is very useful for studies of the reaction mechanism, structural/functional relationships, and drug interactions. We report here a heterologous system for overexpression of human TXAS. The TXAS cDNA was modified by replacing the sequence encoding the first 28 amino acid residues with a CYP17 amino-terminal sequence and by adding a polyhistidine tag sequence prior to the stop codon; the cDNA was inserted into the pCW vector and co-expressed with chaperonins groES and groEL in Escherichia coli. The resulting recombinant protein was purified to electrophoretic homogeneity by affinity, ion exchange, and hydrophobic chromatography. UV-visible absorbance (UV-Vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectra indicate that TXAS has a typical low spin cytochrome P450 heme with an oxygen-based distal ligand. The UV-Vis and EPR spectra of recombinant TXAS were essentially identical to those of TXAS isolated from human platelets, except that a more homogenous EPR spectrum was observed for the recombinant TXAS. The recombinant protein had a heme:protein molar ratio of 0.7:1 and a specific activity of 12 micromol of TXA2/min/mg of protein at 23 degreesC. Furthermore, it catalyzed formation of TXA2, 12-hydroxy-5,8,10-heptadecatrienoic acid, and malondialdehyde in a molar ratio of 0.94:1.0:0.93. Spectral binding titrations showed that bulky heme ligands such as clotrimazole bound strongly to TXAS (Kd approximately 0.5 microM), indicating ample space at the distal face of the heme iron. Analysis of MCD and EPR spectra showed that TXAS was a typical low spin hemoprotein with a proximal thiolate ligand and had a very hydrophobic distal ligand binding domain.

An improved sample packing device for rapid freeze-trap electron paramagnetic resonance spectroscopy kinetic measurements.

A new method has been developed for sample packing in rapid freeze-quench electron paramagnetic resonance spectroscopy (EPR) kinetic experiments. Sample particles freeze-quenched in chilled isopentane are filtered under pressure through a stainless steel funnel attached to an EPR tube fitted with a porous disk at its bottom. Isopentane exits through the porous disk and the sample particles can be transferred essentially quantitatively into the receiving EPR tube. This device provides a more predictable, reproducible, and time-saving method for sample packing, enables use of a wider range of flow velocity, and allows efficient use of valuable reactants.

Cellular regulation of prostaglandin H synthase catalysis.

Prostanoids are a group of potent bioactive lipids produced by oxygenation of arachidonate or one of several related polyunsaturated fatty acids. Cellular prostaglandin biosynthesis is tightly regulated, with a large part of the control exerted at the level of cyclooxygenase catalysis by prostaglandin H synthase (PGHS). The two known isoforms of PGHS have been assigned distinct pathophysiological functions, and their cyclooxygenase activities are subject to differential cellular control. This review considers the contributions to cellular catalytic control of the two PGHS isoforms by intracellular compartmentation, accessory proteins, arachidonate levels, and availability of hydroperoxide activator.

Comparison of structural stabilities of prostaglandin H synthase-1 and -2.

There are two known isoforms of prostaglandin H synthase (PGHS), a key enzyme in the conversion of arachidonic acid to bioactive prostanoids. The "constitutive" isoform, PGHS-1, is thought to have housekeeping functions, and the "inducible" isoform, PGHS-2, has been implicated in cellular responses to cytokines. The two isoforms have high sequence conservation in the cyclooxygenase active site and quite similar crystallographic structures, but differ markedly in their interactions with many cyclooxygenase substrates and inhibitors. We have evaluated the stability of the overall folding, and of the active sites of ovine PGHS-1 and human PGHS-2 using denaturation with guanidinium hydrochloride (GdmHCl). Changes in hydrodynamic and cross-linking properties indicated a dimer --> monomer transition for both isoforms between 0.5 and 2 M GdmHCl; the monomers unfolded at higher GdmHCl levels. Changes in overall secondary and tertiary structure, measured by tryptophan fluorescence and circular dichroism, occurred in two phases for each isoform, with the transition between the phases at 0.2-0.5 M GdmHCl. Disruption of active site functions (cyclooxygenase, peroxidase, and cyclooxygenase inhibitor binding activities) began at GdmHCl levels below 0.2 M. The structural and functional changes were completely reversible up to about 2 M GdmHCl, they were more pronounced at lower protein levels, and they required lower GdmHCl levels for PGHS-2 than for PGHS-1. The results are consistent with a four-state denaturation process for both isoforms: native dimers --> inactive dimers --> compact monomers --> unfolded monomers. The first two steps are reversible for both isoforms; PGHS-2 undergoes the first and last steps more readily than PGHS-1. Thus, the structural stability of PGHS-2, both in the active site regions and in the subunits overall, is distinctly less than that of PGHS-1. These differences in structural stability may contribute to the isoforms' active site ligand selectivity.

Structural characterization of arachidonyl radicals formed by prostaglandin H synthase-2 and prostaglandin H synthase-1 reconstituted with mangano protoporphyrin IX.

A tyrosyl radical generated in the peroxidase cycle of prostaglandin H synthase-1 (PGHS-1) can serve as the initial oxidant for arachidonic acid (AA) in the cyclooxygenase reaction. Peroxides also induce radical formation in prostaglandin H synthase-2 (PGHS-2) and in PGHS-1 reconstituted with mangano protoporphyrin IX (MnPGHS-1), but the EPR spectra of these radicals are distinct from the initial tyrosyl radical in PGHS-1. We have examined the ability of the radicals in PGHS-2 and MnPGHS-1 to oxidize AA, using single-turnover EPR studies. One wide singlet tyrosyl radical with an overall EPR line width of 29-31 gauss (G) was generated by reaction of PGHS-2 with ethyl hydroperoxide. Anaerobic addition of AA to PGHS-2 immediately after formation of this radical led to its disappearance and emergence of an AA radical (AA.) with a 7-line EPR, substantiated by experiments using octadeuterated AA. Subsequent addition of oxygen resulted in regeneration of the tyrosyl radical. In contrast, the peroxide-generated radical (a 21G narrow singlet) in a Y371F PGHS-2 mutant lacking cyclooxygenase activity failed to react with AA. The peroxide-generated radical in MnPGHS-1 exhibited a line width of 36-38G, but was also able to convert AA to an AA. with an EPR spectrum similar to that found with PGHS-2. These results indicate that the peroxide-generated radicals in PGHS-2 and MnPGHS-1 can each serve as immediate oxidants of AA to form the same carbon-centered fatty acid radical that subsequently reacts with oxygen to form a hydroperoxide. The EPR data for the AA-derived radical formed by PGHS-2 and MnPGHS-1 could be accounted for by a planar pentadienyl radical with two strongly interacting beta-protons at C10 of AA. These results support a functional role for peroxide-generated radicals in cyclooxygenase catalysis by both PGHS isoforms and provide important structural characterization of the carbon-centered AA..

Stoichiometry of the interaction of prostaglandin H synthase with substrates.

Prostaglandin H synthase (PGHS) catalyzes both peroxidase and cyclooxygenase reactions. Resolution of several current issues regarding the PGHS catalytic mechanism hinges on the stoichiometry of the reaction of PGHS with hydroperoxide, fatty acid, and oxygen. The dependence of wide-doublet tyrosyl radical accumulation in PGHS isoform 1 on hydroperoxide stoichiometry, has been determined; this catalytically active radical is formed efficiently at stoichiometries