Structural Characterization of Arachidonyl Radicals Formed by Prostaglandin H Synthase-2 and Prostaglandin H Synthase-1 Reconstituted with Mangano Protoporphyrin IX

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Peroxide-generated tyrosyl radicals in both prostaglandin H synthase (PGHS) isozymes have been demonstrated to couple the peroxidase and cyclooxygenase activities by serving as the immediate oxidant for arachidonic acid (AA) in cyclooxygenase catalysis. Acetylation of Ser-530 of PGHS-1 by aspirin abolishes all oxygenase activity and transforms the peroxide-induced tyrosyl radical from a functional 33-35-gauss (G) wide doublet/wide singlet to a 26-G narrow singlet unable to oxidize AA. In contrast, aspirin-treated PGHS-2 (ASA-PGHS-2) no longer forms prostaglandins but retains oxygenase activity forming 11(R)-and 15(R)-hydroperoxyeicosatetraenoic acid and also retains the EPR line-shape of the native peroxide-induced 29 -30-G wide singlet radical. To evaluate the functional role of the wide singlet radical in ASA-PGHS-2, we have examined the ability of this radical to oxidize AA in singleturnover EPR studies. Anaerobic addition of AA to ASA-PGHS-2 immediately after formation of the wide singlet radical generated either a 7-line EPR signal similar to the pentadienyl AA radical obtained in native PGHS-2 or a 26 -28-G singlet radical. These EPR signals could be accounted for by a pentadienyl radical and a strained allyl radical, respectively. Experiments using 11d-AA, 13(R)d-AA, 15d-AA, 13,15d 2 -AA, and octadeuterated AA (d 8 -AA) confirmed that the unpaired electron in the pentadienyl radical is delocalized over C11, C13, and C15. A 6-line EPR radical was observed when 16d 2 -AA was used, indicating only one strongly interacting C16 hydrogen. These results support a functional role for peroxide-generated tyrosyl radicals in lipoxygenase catalysis by ASA-PGHS-2 and also indicate that the AA radical in ASA-PGHS-2 is more constrained than the corresponding radical in native PGHS-2.

Section: Sequential Reaction Of Asa-pghs-2 With Hydroperoxidesupporting

confidence: 80%

“…Single turnover experiments using an anaerobic titrator were performed as previously described (16,17). EPR spectra were recorded at liquid nitrogen temperatures on a Varian E-6 or a Bruker EMX spectrometer (16).…”

Section: Methodsmentioning

confidence: 99%

Structural Characterization of Arachidonyl Radicals Formed by Aspirin-treated Prostaglandin H Synthase-2

Tsai¹,

Palmer²,

Wu³

et al. 2002

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“…As for cyclooxygenase catalysis itself, there have been several proposals for the key enzyme oxidant in the process, including ferrous and perferryl (Compound I) states of the heme, and a tyrosyl radical (11,21,42). Accumulating spectroscopic and kinetic observations for reactions with arachidonate or eicosadienoic acid (23)(24)(25)(26) have now established the tyrosyl radical as the central cyclooxygenase oxidant.…”

Section: Discussionmentioning

confidence: 99%

“…A tyrosyl radical at residue 385 (ovine PGHS-1 numbering) has emerged as a key cyclooxygenase catalytic intermediate in both isoforms (21)(22)(23)(24)(25)(26), and branched-chain reaction mechanisms based on the tyrosyl radical successfully account for many aspects of peroxidase and cyclooxygenase kinetics (15,27,28). However, one perceived deficiency of branched-chain mechanisms has been an inability to predict significant stimulation of cyclooxygenase velocity by cosubstrates without ad hoc assumptions (15,17).…”

mentioning

confidence: 99%

Prostaglandin H Synthase

Bambai

Kulmacz

2000

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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.by guest on May 7, 2018 http://www.jbc.org/ Downloaded from 3 The cyclooxygenase activity of prostaglandin H synthase isoforms 1 and 2 1 is a key control point in the biosynthesis of all prostanoid lipid mediators (1,2). Besides the cyclooxygenase activity, both PGHS-1 and -2 have a heme-dependent peroxidase activity (1). The PGHS peroxidase catalytic cycle resembles that of other heme-dependent peroxidases: the ferric heme of the resting enzyme is oxidized to Intermediate I (Compound I) by reaction with peroxide, and electron-donating cosubstrates complete the peroxidase catalytic cycle by reducing Intermediate I to Compound II and then to resting enzyme (3). Endogenous peroxidase cosubstrates, such as uric acid, are present in cell cytosol (4).Cosubstrates for PGHS peroxidase also have been reported to affect PGHS cyclooxygenase activity, attenuating self-inactivation and increasing the catalytic velocity (5-13). The protective action of cellular reductants greatly increases the number of catalytic turnovers before cyclooxygenase selfinactivation, thereby increasing the capacity for synthesis of potent lipid mediators (14). For its part, the large stimulation of cyclooxygenase velocity by cosubstrate has become a defining characteristic of catalytic behavior and an important criterion for evaluation of potential PGHS reaction mechanisms (11,13,15 (15,27,28). However, one perceived deficiency of branched-chain mechanisms has been an inabili...

“…The initiation process is believed to entail reaction of the hydroperoxide with the heme in the peroxidase site to form an oxidized intermediate, Compound I, which then undergoes an intramolecular redox step to generate a tyrosyl radical in the cyclooxygenase pocket (22). Tyrosyl radicals have been shown to be capable of serving as the initial oxidant in the cyclooxygenase catalytic cycle for both PGHS isoforms (23,24). Because the product of cyclooxygenase catalysis, PGG 2 , is itself a hydroperoxide, cyclooxygenase catalysis in bulk solution is a branched-chain process with feedback acceleration as PGG 2 produced by enzyme activated early on diffuses away to initiate cyclooxygenase catalysis in previously latent enzyme (16).…”

Section: Discussionmentioning

confidence: 99%

Hydroperoxide Dependence and Cooperative Cyclooxygenase Kinetics in Prostaglandin H Synthase-1 and -2

Chen

Pawelek

Kulmacz

1999

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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. The cyclooxygenase activity of prostaglandin H synthase (PGHS)1 catalyzes the oxygenation of arachidonic acid to PGG 2 , a key control step in the biosynthesis of all prostanoids (1). Two isoforms of PGHS are known: PGHS-1 is present at relatively stable levels in many cells, and is considered a housekeeping enzyme, whereas PGHS-2 is absent from most quiescent cells but is rapidly induced in cells responding to cytokines and mitogens (2). Both isoforms are hemoproteins with peroxidase and cyclooxygenase activities (3). Studies with recombinant human PGHS-1 and -2 indicate that the two isoforms have similar peroxidase and cyclooxygenase specific activities (4).Cyclooxygenase synthetic capacity tends to be much greater than the actual prostaglandin synthesis from endogenous fatty acid in vivo, demonstrating that regulation of cyclooxygenase catalysis plays a major role in controlling cellular prostanoid synthesis (5). In this regard, it is particularly interesting that the two isoforms' cyclooxygenase catalytic activities are differentially controlled in several cells where both PGHS-1 and -2 are present (2). In these cells, the large pulse of prostaglandin synthesis that follows cytokine treatment comes from cyclooxygenase catalysis by freshly induced PGHS-2; the considerable PGHS-1 cyclooxygenase capacity in the same cells remains latent unless high levels of arachidonate are added.One possible explanation for this differe...