Studies on the reduction of endogenously generated prostaglandin G2 by prostaglandin H synthase

Prostaglandin H synthase oxidizes arachidonic acid to prostaglandin G2 (PGG2) via its cyclooxygenase activity and reduces PGG2 to prostaglandin H2 by its peroxidase activity. The purpose of this study was to determine if endogenously generated PGG2 is the preferred substrate for the peroxidase compared with exogenous PGG2. Arachidonic acid and varying concentrations of exogenous PGG2 were incubated with ram seminal vesicle microsomes or purified prostaglandin H synthase in the presence of the reducing cosubstrate, aminopyrine. The formation of the aminopyrine cation free radical (AP.+) served as an index of peroxide reduction. The simultaneous addition of PGG2 with arachidonic acid did not alter cyclooxygenase activity of ram seminal vesicle microsomes or the formation of the AP.+. This suggests that the formation of AP.+, catalyzed by the peroxidase, was supported by endogenous endoperoxide formed from arachidonic acid oxidation rather than by the reduction of exogenous PGG2. In addition to the AP.+ assay, the reduction of exogenous versus endogenous PGG2 was studied by using [5,6,8,9,11,12,14,15-2H]arachidonic acid and unlabeled PGG2 as substrates, with gas chromatography-mass spectrometry techniques to measure the amount of reduction of endogenous versus exogenous PGG2. Two distinct results were observed. With ram seminal vesicle microsomes, little reduction of exogenous PGG2 was observed even under conditions in which all of the endogenous PGG2 was reduced. In contrast, studies with purified prostaglandin H synthase showed complete reduction of both exogenous and endogenous PGG2 using similar experimental conditions. Our findings indicate that PGG2 formed by the oxidation of arachidonic acid by prostaglandin H synthase in microsomal membranes is reduced preferentially by prostaglandin H synthase.     

The oxidation of arachidonic acid by the cyclooxygenase activity of purified prostaglandin H synthase: spin trapping of a carbon-centered free radical intermediate

The ESR spin trapping technique was used to study the first detectable radical intermediate in the oxidation of arachidonic acid by purified prostaglandin H synthase. The holoenzyme and the apoenzyme, reconstituted with either hematin or Mn2+ protoporphyrin IX, were investigated. Depending on the different types of enzyme activity present, arachidonic acid was oxidized to at least two free radicals. One of these radicals is thought to be the first ESR detectable radical intermediate in the conversion of arachidonic acid to prostaglandin G2 and was detected previously in incubations of ram seminal vesicle microsomes, which are rich in prostaglandin H synthase. The ESR findings correlated with oxygen incorporation into arachidonic acid and prostaglandin formation, where the spin trap inhibits oxygen incorporation and prostaglandin formation by apparently competing with oxygen for the carbon-centered radical. Substitution of arachidonic acid by octadeuterated (5, 6, 8, 9, 11, 12, 14, 15)-arachidonic acid confirmed that the radical adduct contained arachidonic acid that is bound to the spin trap at one of these eight positions. An attempt was made to explain the apparent time lag between the metabolic activity observed in the oxygraph measurements and the appearance of the trapped radical signals.     

Electron spin resonance investigation of tyrosyl radicals of prostaglandin H synthase. Relation to enzyme catalysis.

We have examined, by low temperature ESR, the protein-derived radicals formed by reaction of purified ram seminal vesicle prostaglandin H synthase (PHS). Upon addition of arachidonic acid or 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP) to PHS reconstituted with Fe(III)-protoporphyrin IX (Fe-PHS) at -12 degrees C, an ESR spectrum was observed at -196 degrees C containing a doublet that rapidly converted into a singlet. These protein-derived radicals were identified as tyrosyl radicals. The addition of a peroxidase substrate, phenol, completely abolished the appearance of the doublet and suppressed the formation of the singlet but did not inhibit eicosanoid formation. Incubation of arachidonic acid with PHS reconstituted with Mn(III)-protoporphyrin IX (Mn-PHS) produced only a broad singlet that exhibited different power saturation behavior than the tyrosyl radicals and decayed more rapidly. This broad singlet does not appear to be a tyrosyl radical. No ESR signals were observed on incubation of PPHP with Mn-PHS, which has cyclooxygenase but not peroxidase activity. Eicosanoid synthesis occurred very rapidly after addition of arachidonic acid and was complete within 1 min. In contrast, the protein-derived radicals appeared at a slower rate and after the addition of the substrate reached maximal levels between 1 and 2 min for Fe-PHS and 4-6 min for Mn-PHS. These results suggest that the observed protein-derived radicals are not catalytically competent intermediates in cyclooxygenase catalysis by either Fe-PHS or Mn-PHS. The peroxidase activity appears to play a major role in the formation of the tyrosyl radicals with Fe-PHS.     

Prostaglandin synthase-catalyzed metabolic activation of some aromatic amines to genotoxic products

Cultured human fibroblasts were incubated with different aromatic amines in the presence of different activation systems and the induction of strand breaks in fibroblast DNA was studied. In the presence of ram seminal vesicle microsomes and arachidonic acid, DNA strand breaks were induced by 2-naphthylamine, 2,4-diaminotoluene and 4-methoxy-m-phenylenediamine. This effect was decreased when the prostaglandin synthase of the ram seminal vesicle microsomes was inhibited. The data suggest that metabolic activation catalyzed by prostaglandin synthase may be of importance in the formation of genotoxic products by certain urinary tract carcinogens.     

Biosynthesis of 5,6-dihydroxyprostaglandin E1 and F1 alpha from 5,6-dihydroxyeicosatrienoic acid by ram seminal vesicles

cis-5(6)Epoxy-cis-8,11,14-eicosatrienoic acid was recently found to be metabolized by ram seminal vesicles to 5-hydroxyprostaglandin I 1 alpha and 5-hydroxyprostaglandin I 1 beta, 5(6)epoxyprostaglandin E1 and 5,6-dihydroxyprostaglandin E1. The epoxide can be hydrolyzed by epoxide hydrolases to 5,6-dihydroxy-8,11,14-eicosatrienoic acid. The latter was incubated with microsomes of ram seminal vesicles for 2 min at 37 degrees C and the polar metabolites were purified by reversed phase HPLC and analyzed by capillary column gas chromatography-mass spectrometry. The major metabolite was identified as 5,6-dihydroxyprostaglandin F 1 alpha. In the presence of glutathione (1 mM), 5,6-dihydroxyprostaglandin E1 was also formed. The 3H-labelled vicinal diol and the 3H-labelled epoxide were metabolized to polar products to a similar extent, but the formation of prostaglandin E compounds in the presence of glutathione was lower from the diol than from the epoxide or from arachidonic acid. The likely prostaglandin endoperoxide intermediates in the metabolism of the diol (5,6-dihydroxyprostaglandin G1 and 5,6-dihydroxyprostaglandin H1) thus appear to be less prone to be isomerized to prostaglandin E compounds than prostaglandins G2 and H2 and their 5(6)epoxy counterparts. 5(6)Epoxyprostaglandin E1 and 5,6-dihydroxyprostaglandin E1 can be chemically transformed into 5,6-dihydroxyprostaglandin B1. The latter can be analyzed by HPLC or by mass fragmentography, and a simple chemical synthesis of 5,6-dihydroxyprostaglandin B1 from prostaglandin E2 is described.     

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.     

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.     

The formation of aminopyrine cation radical by the peroxidase activity of prostaglandin H synthase and subsequent reactions of the radical

The oxidation of aminopyrine to an aminopyrine cation radical was investigated using a solubilized microsomal preparation or prostaglandin H synthase purified from ram seminal vesicles. Aminopyrine was oxidized to an aminopyrine cation radical in the presence of arachidonic acid, hydrogen peroxide, t-butyl hydroperoxide or 15-hydroperoxyarachidonic acid. Highly purified prostaglandin H synthase, which processes both cyclo-oxygenase and hydroperoxidase activity, oxidized aminopyrine to the free radical. Purified prostaglandin H synthase reconstituted with Mn2+ protoporphyrin IX, which processes only cyclo-oxygenase activity, did not catalyze the formation of the aminopyrine free radical. Aminopyrine stimulated the reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid to 15-hydroxy-5,8,11-13-eicosatetraenoic acid. Approximately 1 molecule of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid was reduced for every 2 molecules of aminopyrine free radical formed, giving a stoichiometry of 1:2. The decay of the aminopyrine radical obeyed second-order kinetics. These results support the proposed mechanism in which aminopyrine is oxidized by prostaglandin H synthase hydroperoxidase to the aminopyrine free radical, which then disproportionates to the iminium cation. The iminium cation is further hydrolyzed to the demethylated amine and formaldehyde. Glutathione reduced the aminopyrine radical to aminopyrine with the concomitant oxidation of GSH to its thiyl radical as detected by ESR of the glutathione thiyl radical adduct.     

Prostaglandin H synthase: distinct binding sites for cyclooxygenase and peroxidase substrates

Prostaglandin H synthase has two distinct catalytic activities: a cyclooxygenase activity that forms prostaglandin G2 from arachidonic acid; and a peroxidase activity that reduces prostaglandin G2 to prostaglandin H2. Lipid hydroperoxides, such as prostaglandin G2, also initiate the cyclooxygenase reaction, probably via peroxidase reaction cycle enzyme intermediates. The relation between the binding sites for lipid substrates of the two activities was investigated with an analysis of the effects of arachidonic and docosahexaenoic acids on the reaction kinetics of the peroxidase activity, and their effects on the ability of a lipid hydroperoxide to initiate the cyclooxygenase reaction. The cyclooxygenase activity of pure ovine synthase was found to have an apparent Km value for arachidonate of 5.3 microM and a Ki value (competetive inhibitor) for docosahexaenoate of 5.2 microM. When present at 20 microM neither fatty acid had a significant effect on the apparent Km value of the peroxidase for 15-hydroperoxyeicosatetraenoic acid: the values were 7.6 microM in the absence of docosahexaenoic acid and 5.9 microM in its presence, and (using aspirin-treated synthase) 13.7 microM in the absence of arachidonic acid and 15.7 microM in its presence. Over a range of 1 to 110 microM the level of arachidonate had no significant effect on the initiation of the cyclooxygenase reaction by 15-hydroperoxyeicosatetraenoic acid. The inability of either arachidonic acid or docosahexaenoic acid to interfere with the interaction between the peroxidase and lipid hydroperoxides indicates that the cyclooxygenase and peroxidase activities of prostaglandin H synthase have distinct binding sites for their lipid substrates.     

The free radical formed during the hydroperoxide-mediated deactivation of ram seminal vesicles is hemoprotein-derived

Prostaglandin synthase is a multi-enzyme complex which catalyzes the oxygenation of arachidonic acid to the various prostaglandins. During the oxygenation, the enzyme is self-deactivated and, on the basis of ESR data, it has been proposed to form a self-destructive free radical. The free radical was suggested to form from the oxygen lost from prostaglandin G2 during its reduction to prostaglandin H2, and the destructive species was therefore thought to be an oxygen-centered free radical, tentatively identified as the hydroxy radical. We have reinvestigated this ESR signal (g = 2.005) and have concluded, with the aid of the known ESR parameters for the hydroxy and other oxygen-centered free radicals, that the free radical formed during the oxygenation is neither a hydroxy nor any known oxygen-centred radical. Prostaglandin synthase is thought to be a hemoprotein, so this unknown ESR signal was compared with the previously observed free radical formed by the reaction of H2O2 with methemoglobin. This comparison indicates that the free radical formed by the reaction of prostaglandin G2 with ram seminal vesicles is hemoprotein-derived and may be formed by the oxidation of an amino acid(s) located near the iron of the heme.     

Regional distribution of prostaglandin endoperoxide synthase studied by enzyme-linked immunoassay using monoclonal antibodies

Prostaglandin endoperoxide synthase transforms arachidonic acid to prostaglandin H2 via prostaglandin G2. The enzyme purified from bovine vesicular gland was given to mice as antigen, and monoclonal antibodies were raised by the hybridoma technique. Two species of the monoclonal antibody recognizing different sites of the enzyme were utilized to establish a peroxidase-linked immunoassay of prostaglandin endoperoxide synthase. Fab' fragment of one of the antibodies was prepared and conjugated to horseradish peroxidase. The conjugate was then bound to prostaglandin endoperoxide synthase, and the labeled enzyme was precipitated by the addition of the other antibody. The peroxidase activity of the immunoprecipitate correlated linearly with the amount of prostaglandin endoperoxide synthase. This sensitive and convenient method to determine the enzyme amount rather than the enzyme activity was utilized to extensively screen the amount of prostaglandin endoperoxide synthase in various bovine tissues. In addition to vesicular gland, platelets and kidney medulla previously known as rich enzyme sources, the immunoenzymometric assay demonstrated a high content of the enzyme in various parts of alimentary tract and a low but significant amount of enzyme in some parts of brain.     

A test for the intermediacy of 11-hydroperoxyeicosa-5,8,12,14-tetraenoic acid [11-HPETE] in prostaglandin biosynthesis

11-Hydroperoxy-eicosa-5,8,12,14-tetraenoic acid [11-HPETE] was prepared by chromatographic separation of the hydroperoxides formed from the singlet oxygen oxidation of arachidonic acid [20:4]. 1-[¹⁴C]-HPETE was incubated with prostaglandin endoperoxide synthetase preparations from ram seminal vesicles. No prostaglandins products deriving from 11-HPETE were detected in any of the incubations. 11-Hydroxy-eicosa-5,8,12,14-tetraenoic acid [11-HETE], formed by the action of the hydroperoxidase component of prostaglandin endoperoxidase synthetase was the major product formed. The peroxidase activity was absolutely dependent on epinephrine and was stimulated by hematin. 11-HPETE does not appreciably effect the extent of conversion of arachidonic acid into prostaglandin.     

Isolation and biosynthesis of 20-hydroxyprostaglandins E1 and E2 in ram seminal fluid

Ram semen was found to contain 20-hydroxyprostaglandin E1 and 20-hydroxyprostaglandin E2. The relative amounts of the two compounds were almost equal, although ram semen contained at least 10 times more prostaglandin E1 than prostaglandin E2. The accessory genital glands of the ram were analyzed for their capacity to metabolize [14C]arachidonic acid to prostaglandins. Biosynthesis of prostaglandins was only found in microsomes of the mucosa of the ampulla of vas deferens and in microsomes of the vesicular glands. Ram vesicular glands and the ampulla of vas deferens were also found to contain the two 20-hydroxylated E prostaglandins. Microsomes of ram vesicular glands and NADPH metabolized exogenous prostaglandin E2 to 20-hydroxyprostaglandin E2 albeit in low yields. Prostaglandin E2 appeared to be a better substrate than prostaglandin E1. Microsomes of human seminal vesicles and NADPH metabolized exogenous prostaglandin E2 to 19-hydroxyprostaglandin E2. The results show that 19- and 20-hydroxylation of prostaglandins occurs in human and ram seminal vesicles, respectively, and possibly also in the ampulla of vas deferens of the ram. The ram and human enzymes specifically hydroxylated the terminal and the penultimate carbon of prostaglandin E2, respectively.     

Higher oxidation states of prostaglandin H synthase. EPR study of a transient tyrosyl radical in the enzyme during the peroxidase reaction

Purified prostaglandin H synthase (EC 1.14.99.1), reconstituted with hemin, was reacted with substrates of the cyclooxygenase and peroxidase reaction. The resulting EPR spectra were measured below 90 K. Arachidonic acid, added under anaerobic conditions, did not change the EPR spectrum of the native enzyme due to high-spin ferric heme. Arachidonic acid with O2, as well as prostaglandin G2 or H2O2, decreased the spectrum of the native enzyme and concomitantly a doublet signal at g = 2.005 was formed with maximal intensity of 0.35 spins/enzyme and a half-life of less than 20 s at -12 degrees C. From the conditions for the formation and the effect of inhibitors, this doublet signal was assigned to an enzyme intermediate of the peroxidase reaction, namely a higher oxidation state. The doublet signal with characteristic hyperfine structure was nearly identical to the signal of the tyrosyl radical in ribonucleotide reductase (EC 1.17.4.1). Hence the signal of prostaglandin H synthase was assigned to a tyrosyl radical. Electronic spectra as well as decreased power saturation of the tyrosyl radical signal indicated heme in its ferryl state which coupled to the tyrosyl radical weakly. [FeIVO(protoporphyrin IX)]...Tyr+. was suggested as the structure of this two-electron oxidized state of the enzyme. A hypothetical role for the tyrosyl radical could be the abstraction of a hydrogen at C-13 of arachidonic acid which is assumed to be the initial step of the cyclooxygenase reaction.     

The formation of styrene glutathione adducts catalyzed by prostaglandin H synthase. A possible new mechanism for the formation of glutathione conjugates

The metabolism of styrene by prostaglandin hydroperoxidase and horseradish peroxidase was examined. Ram seminal vesicle microsomes in the presence of arachidonic acid or hydrogen peroxide and glutathione converted styrene to glutathione adducts. Neither styrene 7,8-oxide nor styrene glycol was detected as a product in the incubation. Also, the addition of styrene 7,8-oxide and glutathione to ram seminal vesicle microsomes did not yield styrene glutathione adducts. The peroxidase-generated styrene glutathione adducts were isolated by high pressure liquid chromatography and characterized by NMR and tandem mass spectrometry as a mixture of (2R)- and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione. (1R)- and (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione were not formed by the peroxidase system. The addition of phenol or aminopyrine to incubations, which greatly enhances the oxidation of glutathione to a thiyl radical by peroxidases, increased the formation of styrene glutathione adducts. We propose a new mechanism for the formation of glutathione adducts that is independent of epoxide formation but dependent on the initial oxidation of glutathione to a thiyl radical by the peroxidase, and the subsequent reaction of the thiyl radical with a suitable substrate, such as styrene.     

A free radical mechanism of prostaglandin synthase-dependent aminopyrine demethylation

The mechanism of prostaglandin synthase-dependent N-dealkylation has been investigated using an enzyme preparation derived from ram seminal vesicles. Incubation of an N-alkyl substrate, aminopyrine, with enzyme and arachidonic acid, 15-hydroperoxyarachidonic acid, or tert-butyl hydroperoxide resulted in the formation of the transient aminopyrine free radical species. Formation of this radical species, which was detected by electron paramagnetic resonance spectroscopy and/or absorbance at 580 nm, was maximal approximately 30 s following initiation of the reaction and declined thereafter. Free radical formation corresponded closely with formaldehyde formation in this system, in terms of dependence upon substrate and cofactor concentration, as well as in terms of time course. Both aminopyrine free radical and formaldehyde formation were inhibited by indomethacin and flufenamic acid, inhibitors of prostaglandin synthase. The results suggest that the aminopyrine free radical is an intermediate in the prostaglandin synthase-dependent aminopyrine N-demethylase pathway. The aminopyrine free radical electron paramagnetic resonance spectrum revealed that this species is a one-electron oxidized cation radical of the parent compound. A reaction mechanism has been proposed in which aminopyrine undergoes two sequential one-electron oxidations to an iminium cation, which is then hydrolyzed to the demethylated amine and formaldehyde. Accordingly, the oxygen atom of the aldehyde product is derived from neither molecular nor hydroperoxide oxygen, but from water.     

The formation of stable fatty acid substrate complexes in prostaglandin H(2) synthase-1

We have developed a protocol to purify apo-ovine (o) prostaglandin endoperoxide H(2) synthase-1 (PGHS-1) to homogeneity from ram seminal vesicles. The resulting apo enzyme can then be reconstituted with Co(3+)-protoporphyrin IX instead of Fe(3+)-protoporphyrin IX to produce a native-like, but functionally inert, enzyme suitable for the production of enzyme:fatty acid substrate complexes for biophysical characterization. Co(3+)-protoporphyrin IX reconstituted oPGHS-1 (Co(3+)-oPGHS-1) displays a Soret band at 426 nm that shifts to 406 nm upon reduction. This behavior is similar to that of cobalt-reconstituted horseradish peroxidase and myoglobin and suggests, along with resonance Raman spectroscopy, that the Co(3+)-protoporphyrin IX group is one in a six-coordinate, cobalt(III) state. However, Co(3+)-oPGHS-1 does not display cyclooxygenase or peroxidase activity, nor does the enzyme produce prostaglandin products when incubated with [1-(14)C]arachidonic acid. The cocrystallization of Co(3+)-oPGHS-1 and the substrate arachidonic acid (AA) has been achieved using sodium citrate as the precipitant in the presence of the nonionic detergent N-octyl-beta-d-glucopyranoside. Crystals are hexagonal, belonging to the space group P6(5)22, with cell dimensions of a = b = 181.69 A and c = 103.74 A, and a monomer in the asymmetric unit. GC-MS analysis of dissolved crystals indicates that unoxidized AA is bound within the crystals.     

Higher oxidation states of prostaglandin H synthase. Rapid electronic spectroscopy detected two spectral intermediates during the peroxidase reaction with prostaglandin G2

The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1 degree C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1 = 1.4 x 10(7) M-1 s-1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+.FeIVO]. Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2 = 65 s-1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome-c peroxidase, was assigned to a two-electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+. which was formed by an intramolecular electron transfer from tyrosine to the porphyrin-pi-cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction.     

Prostaglandin endoperoxide synthase substituted with manganese protoporphyrin IX. Formation of a higher oxidation state and its relation to cyclooxygenase reaction.

The heme in prostaglandin endoperoxide synthase (PGH synthase) was substituted with Mn(III)-protoporphyrin IX. The resulting enzyme, Mn-PGH synthase, showed full cyclooxygenase activity but only 0.9% of the peroxidase activity of the native iron enzyme. During the reaction with exogenous or endogenously produced hydroperoxides, a spectral intermediate of Mn-PGH synthase was observed. The electronic absorption bands of the resting enzyme at 376, 472, and 561 nm decreased, and the intermediate's bands at 417, around 513, and 625 nm appeared. The rate constant of the formation of the intermediate was about 10(4) M-1.s-1 at 22 degrees C, three orders of magnitude lower than with the iron enzyme. Spectral properties, conditions of formation, and the suppressed formation in the presence of electron donors provide evidence for a higher oxidation state of Mn-PGH synthase, tentatively a Mn(IV) species. This species was assigned to an intermediate in the peroxidase reaction of Mn-PGH synthase, the low activity of which was explained by the rate-limiting slow reaction of Mn-PGH synthase with hydroperoxides. The findings and interpretation are consistent with the published properties of other manganese-substituted peroxidases. Although the cyclooxygenase activity was similar to that of Fe-PGH synthase, the cyclooxygenase reaction of Mn-PGH synthase showed distinct differences in comparison with Fe-PGH synthase. A longer activation phase was observed which resembled the time course of the formation of the higher oxidation state. Glutathione peroxidase with glutathione, a hydroperoxide-scavenging system, inhibited the cyclooxygenase of Mn-PGH synthase at concentrations where the activity of Fe-PGH synthase was not affected. It is demonstrated that Mn-PGH synthase requires higher concentrations of hydroperoxides for the activation of the cyclooxygenase. These findings suggest that the substitution of iron with manganese in PGH synthase does not change the mechanism of the enzyme. The main difference is the much lower rate of the reaction with hydroperoxides which affects both the peroxidase activity and the hydroperoxide-dependent activation of the cyclooxygenase. A reaction scheme for Mn-PGH synthase is proposed analogous to that suggested for Fe-PGH synthase (Karthein, R., Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 313-320).     

Stereoselectivity of the epoxidation of 7,8-dihydrobenzo[a]pyrene by prostaglandin H synthase and cytochrome P-450 determined by the identification of polyguanylic acid adducts

The stereoselectivity of the oxidation of 7,8-dihydrobenzo[a]pyrene (H2BP) to 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (H4BP-epoxide) by prostaglandin H (PGH) synthase and cytochrome P-450 has been studied using microsomal preparations from ram seminal vesicles and rat liver. Incubations were performed in the presence of polyguanylic acid and the adducts formed between H4BP-epoxide and guanosine were isolated following the recovery and hydrolysis of the poly(G). When (+/-)-H4BP-epoxide was reacted with poly(G), four diastereomeric adducts were formed by the cis and trans addition of the exocyclic amino group of guanine to the benzylic carbon of the epoxide enantiomers. Each diastereomer was identified by a combination of ultraviolet, nuclear magnetic resonance, circular dichroism, and mass spectroscopy. Under comparable conditions, ram seminal vesicle microsomes in the presence of arachidonic acid triggered the binding of H2BP to poly(G) to a greater extent than rat liver microsomes from untreated and phenobarbital- and methylcholanthrene pretreated animals in the presence of NADPH. Quantitation of the (-)-cis- and (+)-cis-guanosine adducts revealed the degree of stereoselectivity of epoxidation. The ratio of (-)/(+) adducts was 54:46 for PGH synthase and 89:11 (control), 62:38 (phenobarbital), and 69:31 (methylcholanthrene) for cytochrome P-450-catalyzed reactions. PGH synthase catalyzed the epoxidation of H2BP with little or no stereoselectivity in contrast to cytochrome P-450. The utility of the poly(G) binding technique for the elucidation of the stereoselective generation of chiral electrophiles is discussed along with the mechanistic implications of the results.