Reaction of Prostaglandin Endoperoxide Synthase with cis,cis-Eicosa-11,14-dienoic Acid*

Vasilij Koshkin and H. Brian DunfordDagger

From the Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The pre-steady-state phase of the oxygenase reaction of prostaglandin endoperoxide synthase with cis,cis-eicosa-11,14-dienoic acid has been studied using stopped flow techniques. Because some intermediate forms of prostaglandin endoperoxide synthase are spectrally indistinguishable, the enzyme and substrate transformations were monitored in parallel to simplify the interpretation of the kinetics. Over a wide range of conditions, the formation of the enzyme intermediate II, the form of compound I containing the tyrosyl radical, precedes substrate oxidation. This result supports the occurrence of a unimolecular conversion of compound I into intermediate II. Furthermore, the rate of intermediate II formation was stimulated by increased concentration of dienoic acid, perhaps because of increased occupation of the fatty acid binding site. The importance of the unimolecular formation of intermediate II was confirmed by simulated kinetics of the oxygenase reaction. These results provide evidence that intermediate II is the primary oxidant in the reaction of prostaglandin synthase with the dienoic acid, as it is with arachidonic acid.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Prostaglandin H endoperoxide synthase (PGH synthase)1 catalyzes the first step in the biosynthesis of prostaglandins. This step consists of two reactions: the conversion of arachidonic acid into prostaglandin G2 (cyclooxygenase reaction) and the reduction of prostaglandin G2 to prostaglandin H2 (peroxidase reaction) (1-3). Extensive investigation of these reactions has revealed enzymatic intermediates analogous to the classic peroxidase compounds I and II as well as free radical forms of the enzyme (4-6). Two reaction mechanisms have been proposed for this system, differing first of all in relation to the initial events of the cyclooxygenase reaction. The branched chain mechanism considers the tyrosyl radical of PGH synthase, formed from compound I by the intramolecular electron transfer process (Porph·+ FeIVdouble bond O TyrOH right-arrow Porph FeIVdouble bond O TyrO· + H+), as the primary oxidant that abstracts a hydrogen atom from the fatty acid substrate (6, 7). The species Porph FeIVdouble bond O TyrO·is referred to as intermediate II (6). An alternative tightly coupled mechanism has, in the past, assigned this role to a conventional compound I (8, 9). The choice between these possibilities is complicated by the fact that intermediate II and compound II have the same oxidation state of the heme and, consequently, the same optical spectra. EPR spectroscopy, which in principle can detect the free radical intermediate, is unable to follow the reaction on a millisecond time scale, typical for enzyme conversion. In such a situation, parallel kinetic monitoring of both enzyme and substrate behavior could be useful.

cis,cis-Eicosa-11,14-dienoic acid (20:2) has been used in previous studies because it is the only PGH synthase substrate producing easily measurable conjugated diene products that are readily detected spectrophotometrically (10-15). Another advantage of this substrate is that the reaction is relatively slow and can be measured using micromolar concentrations of enzyme. This provides a rare opportunity to observe the enzyme and substrate transformations in parallel, which could give additional information about the PGH synthase reaction mechanism, compared with results obtained with its natural substrate arachidonic acid.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials were purchased from the following sources. cis,cis-Eicosa-11,14-dienoic acid, hematin, diethyldithiocarbamate were from Sigma; phenol was from British Drug House; DE-53 ion exchange chromatography gel was from Whatman; and Tween 20 was from J. T. Baker Chemical Co.

PGH synthase was isolated from ram seminal vesicles microsomes using the method of Marnett et al. (16), modified by MacDonald and Dunford (17). Cyclooxygenase activity was measured with a Clark-type polarographic electrode by monitoring oxygen consumption in the reaction of arachidonic acid with PGH synthase in 0.1 M phosphate buffer, pH 8.0, containing 200 µM arachidonic acid, 1 µM hematin, and 1 mM phenol. Oxygen calibration was performed according to Robinson and Cooper (18), and protein content was measured by the Bio-Rad protein assay using bovine serum albumin as a standard. Preparations of PGH synthase contained 30-50% holoenzyme and consumed 20-40 µmol of oxygen/min/mg of protein; for kinetic measurements, they were reconstituted with the appropriate amount of hematin (19). The kinetic experiments were conducted on an Applied Photophysics SX.17MV (Micro-Volume) stopped flow reaction analyzer. Parallel kinetic runs at 234 nm (absorption peak of conjugated diene products), 414 nm (isosbestic point between compound I and intermediate II), and 426 nm (isosbestic point between compound I and resting enzyme) were performed at 25 °C in 0.1 M phosphate buffer, pH 8.0, to monitor oxidation of 20:2 and formation of compound I and compound I-tyrosyl radical plus compound II. The amount of 20:2 hydroperoxide that always contaminates 20:2 preparations and initiates the oxygenase reaction was regulated either by reduction with immobilized borohydride (20) or by oxidation of 20:2 with oxygen (12) and measured by absorption at 234 nm (21). Molar absorptivities of conjugated diene products, PGH synthase, and its intermediates were taken from earlier studies (21-23). Removal of diethyldithiocarbamate (DDC), used in the enzyme isolation as a stabilizer, was performed, when necessary, by passing the enzyme solution through Sephadex G-25 (medium) preequilibrated with 0.1 M phosphate buffer, pH 8.0, containing 30% glycerol (24). Data were analyzed using a stopped flow reaction analyser kinetic software and a pro Fit data analysis program (Cherwell Scientific, UK).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

The spontaneous oxygenase reaction was studied using 0.75 µM PGH synthase and 10-170 µM 20:2. Typical kinetic traces of the initial phase at different concentrations of 20:2 and 20:2 hydroperoxide are shown in Fig. 1. The common feature of all these experiments is the almost simultaneous formation of compound I and intermediate II and considerable delay in appearance of conjugated diene products. Because addition of oxygen to the fatty acid radical is very fast, formation of the conjugated diene products reflects the rate of hydrogen atom abstraction from 20:2 by PGH synthase.


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Fig. 1.   Kinetics of interaction of PGH synthase (0.75 µM) and 20:2 (44 µM, panel A and 172 µM, panel B). Formation of reaction products was monitored at 234 nm (absorption peak of conjugated dienes), formation of compound I at 414 nm (isosbestic point between compound I and intermediate II), and formation of intermediate II at 426 nm (isosbestic point between resting enzyme and compound I). Dotted lines represent original kinetic traces, and solid lines represent traces averaged over five successive points (total number of recorded points = 400).

The formation of intermediate II and oxidation of 20:2 at different concentrations of 20:2 and constant concentration of 20:2 hydroperoxide is shown in Fig. 2. An increase in 20:2 concentration stimulates both formation of intermediate II and oxidation of 20:2, but again, transformation of substrate noticeably lags the enzyme conversion.


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Fig. 2.   Influence of [20:2] on the formation of intermediate II (solid lines) and oxidation of 20:2 (dotted lines). The reaction mixture contained 0.75 µM PGH synthase, 1.9 µM 20:2 hydroperoxide, and the following concentrations of 20:2: 44.7 µM (traces marked 1), 88.4 µM (traces marked 2), and 131.5 µM (traces marked 3). Kinetic traces consist of averages of each successive five points (total number of recorded points = 400).

The reaction of PGH synthase with 20:2 was performed under single turnover conditions using an approximately equimolar amount of 20:2 hydroperoxide to prevent the reaction of excess hydroperoxide with the compound I to produce compound II (19, 25). Kinetic traces for the system containing 0.77 µM PGH synthase, 0.83 µM 20:2 hydroperoxide, and 9 µM 20:2 are shown in Fig. 3. The results demonstrate that reaction of equimolar amounts of PGH synthase and hydroperoxide leads to formation of intermediate II before the appearance of newly formed hydroperoxide. The molar absorptivity of conjugated dienes at 234 nm is 24 mM-1 cm-1 (21), and the difference in molar absorptivity between intermediate II and resting enzyme at 426 nm is about 17 mM-1 cm-1 (23). Therefore, the absorbance traces can be compared on an approximately equimolar basis.


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Fig. 3.   Kinetics of interaction of PGH synthase and 20:2 at stoichiometric level of 20:2 hydroperoxide. PGH synthase, 0.77 µM; hydroperoxide of 20:2, 0.83 µM; 20:2, 9 µM. Formation of reaction products was monitored at 234 nm (absorption peak of conjugated dienes), formation of compound I at 414 nm (isosbestic point between compound I and intermediate II), and formation of intermediate II at 426 nm (isosbestic point between resting enzyme and compound I). Dotted lines represent original kinetic traces, and solid lines represent traces averaged over five successive points.

DDC is commonly used as a stabilizer during purification and storage of PGH synthase. Under our conditions, it was present in the final mixture in micromolar concentrations, and its reducing action could result in formation of compound II (24). This possibility was tested by performing the reaction at different levels of DDC. The kinetics of appearance of compoundII/intermediate II were fitted to a single exponential function, and kobs values obtained in this way were plotted against [DDC] (Fig. 4). This plot indicates that both DDC-dependent and -independent reactions contribute to the observed reactions. The DDC-dependent part corresponds to reduction of compound I to compound II, whereas the DDC-independent part corresponds to the spontaneous conversion of compound I to intermediate II.


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Fig. 4.   Effect of DDC on intermediate II formation. kobs values were found from exponential fits of kinetic traces of intermediate II formation at 426 nm. Reaction conditions: PGH synthase, 0.73 µM; 20:2 hydroperoxide, 0.86 µM; 20:2, 4.2 µM; DDC 3.1, 6.8, and 11.0 µM.

To achieve greater clarity on the role of DDC, it was replaced by glycerol as stabilizing agent. This resulted in a considerable decrease of the rate of change in absorbance. However, the lag between the kinetics of appearance of intermediate II and oxidation of 20:2 remained. As a typical example Fig. 5 shows kinetic traces for the reaction of PGH synthase with 1.3 equivalents of 20:2 hydroperoxide and 5 equivalents of 20:2 in a DDC-free medium.


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Fig. 5.   Kinetics of interaction of PGH synthase and 20:2 in DDC-free medium at stoichiometric level of 20:2 hydroperoxide. PGH synthase, 0.76 µM; hydroperoxide of 20:2, 0.97 µM; 20:2, 4 µM. Formation of reaction products was monitored at 234 nm (absorption peak of conjugated dienes), formation of compound I at 414 nm (isosbestic point between compound I and intermediate II), and formation of intermediate II at 426 nm (isosbestic point between resting enzyme and compound I). Dotted lines represent original kinetic traces, and solid lines represent traces averaged over five successive points.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

A published mechanism of the oxygenase reaction of PGH synthase (E) with 20:2 (AH) includes the following initial steps up to the rate-limiting formation of eicosadienoyl radical (12-14),



<UP>E + AOOH → E-I + AOH</UP> (Reaction 1)
<UP>E-I + AH → E-II + A<SUP>⋅</SUP></UP> (Reaction 2)
<UP><SC>Scheme</SC> 1</UP>
where E-I is compound I and E-II is compound II. The initiation role of compound I in fatty acid oxidation, proposed by this scheme, disagrees with the observed asynchronous oxidation AH and formation of E-II. Possible alternatives, based on the unimolecular transformation of E-I into intermediate II, E-I-Tyr·, which contains the same number of oxidizing equivalents as compound I (6), or reaction of E-I with a second molecule of hydroperoxide (19), are shown below.
<UP>E + AOOH → E-I + AOH</UP> (Reaction 1)
<UP>E-I → E-I-Tyr<SUP>⋅</SUP></UP> (Reaction 3)
<UP>E-I-Tyr<SUP>⋅</SUP> + AH → E-II + A<SUP>⋅</SUP></UP> (Reaction 4)
<UP><SC>Scheme</SC> 2</UP>
<UP>E + AOOH → E-I + AOH</UP> (Reaction 1)
<UP>E-I + AOOH → E-I-Tyr<SUP>⋅</SUP> + AOOH</UP> (Reaction 5)
<UP>E-I-Tyr<SUP>⋅</SUP> + AH → E-II + A<SUP>⋅</SUP></UP> (Reaction 4)
<UP><SC>Scheme</SC> 3</UP>
Reaction 5 in Scheme 3 is the sum of the following two consecutive reactions.
<UP>E-I + AOOH → E-II + AOO<SUP>⋅</SUP></UP> (Reaction 6)
<UP>E-II + AOO<SUP>⋅</SUP> → E-I-Tyr<SUP>⋅</SUP> + AOOH</UP> (Reaction 7)
<UP><SC>Scheme</SC> 4</UP>
To determine the rate constant for compound I formation (Reaction 1) kinetic traces at 414 nm were obtained over a range of concentrations of 20:2 hydroperoxide. Initial parts of these curves were fitted to a single exponential function, and the resultant kobs values were plotted against [20:2 hydroperoxide]. The plot (Fig. 6) gives kapp = 2.9 × 107 M-1 s-1, which is close to the rate constants for other similar hydroperoxides. The rate constants for reactions (Reactions 3) were estimated from the literature (6, 12, 19, 26), and the following values were used in the simulations: for Scheme 2, k3 = 65 s-1, k4 = 3 × 105 M-1 s-1; for Scheme 3, k5 = 3 × 106 M-1 s-1, k4 = 3 × 105 M-1 s-1.


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Fig. 6.   Plot of kobs versus [20:2-OOH] for compound I formation in DDC (panel A) and glycerol (panel B) containing medium. kapp = 3 × 107 M-1 s-1 (panel A) and 2.5 × 107 M-1 s-1 (panel B).


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Fig. 7.   Experimental and simulated (according to Schemes 2 and 3) kinetics of initial phase of the reaction PGH synthase and 20:2. The solid line represents formation of (E-I-Tyr·+ E-II), and the dotted line represents consumption of 20:2. Initial conditions were as follows: PGH synthase, 0.75 µM; 20:2 and 20:2 hydroperoxide, 19 and 5 µM, respectively (panels A), or 21 and 0.9 µM (panels B).

The behavior of these reaction schemes in relation to 20:2 consumption and production of intermediate II and compound II was tested with a Monte-Carlo method using a CKS simulation program (IBM). Fig. 7 demonstrates experimental and simulated kinetics, using the above rate constants for initiation of the oxygenase reaction with 1.2 or 6.7 eq of 20: 2 hydroperoxide. It shows that the asynchronous oxidation of substrate and appearance of intermediate II at excess of hydroperoxide can be qualitatively predicted by both Schemes 2 and 3, but for a stoichiometric level of hydroperoxide, the rate of oxidation of 20:2 is far too small according to Scheme 3.

Reactions of compound I of PGH synthase are of crucial importance for understanding the catalytic mechanism of this enzyme. Since compound I can convert into the spectrally indistinguishable compound I-tyrosyl radical and compound II, the interpretation of the kinetics of compound I decay becomes difficult. Originally, the enzyme product of compound I decay in the oxygenase reaction of PGH synthase with 20:2 was interpreted as production of compound II, based on the fact that conversion of compound I was a function of fatty acid concentration, indicating a bimolecular reaction. This led to the proposal of Reaction 2 (12) with a delayed formation of the tyrosyl radical in Reactions 6 and 7 (19). However, when both participants of the reaction are monitored, it is apparent that appearance of intermediate II precedes oxidation of fatty acid, though both reactions are functions of the concentration of 20:2. This suggests that dependence of intermediate II production on the concentration of 20:2 is an indirect effect. The stimulation of intermediate II formation by 20:2 could be the result of increased occupation of the fatty acid binding site. This could be the basis of a switching mechanism which would favor initiation of cyclooxygenase cycle and prevent conversion of compound I to compound II when peroxidase reducing substrates are present.

In this light, conversion of compound I into compound II/intermediate II in the reaction of PGH synthase with 20:2 could be caused by one the following: (a) reduction of compound I by DDC used as stabilizing agent (24); (b) reaction of compound I with a second molecule of hydroperoxide (19); or (c) intramolecular electron transfer from Tyr-385 to the porphyrin pi -cation radical (6).

Participation of DDC in intermediate II formation was excluded by the results obtained in a DDC-free medium. The relative significance of bimolecular and unimolecular paths for formation of intermediate II (reasons b and c) apparently depends on the level of hydroperoxide. However, the presence of a large excess of hydroperoxide in vivo appears unlikely, which leaves the intramolecular electron transfer as the most likely route to generate the tyrosyl radical.

From the crystal structure, the arachidonic acid binding site was shown to be located on the proximal side of the heme (27). The role of the Tyr-385 radical in the oxidation of arachidonic acid is now firmly established (28). From the present results, it would appear that the initiation of the oxidation of the eicosadienoic acid proceeds by a similar mechanism.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 403-492-3818; Fax: 403-492-8231; E-mail: Brian.Dunford{at}ualberta.ca.

1 The abbreviations used are: PGH synthase, prostaglandin endoperoxide synthase; Porph, protoporphyrin IX; TyrOH, tyrosyl residue; 20:2, cis,cis-eicosa-11,14-dienoic acid; DDC, diethyldithiocarbamate.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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