Purification and characterisation of prostaglandin endoperoxide synthetase from sheep vesicular glands

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...

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“…3 and 4). (10)(11)(12)(13). These large apparent increases in cyclooxygenase velocity must be reconsidered in light of the current results.…”

Section: Discussionmentioning

confidence: 99%

Prostaglandin H Synthase

Bambai

Kulmacz

2000

Journal of Biological Chemistry

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“…This would tend to extend PGG 2 generation over a longer time. Indeed, the COX reaction in cells provided excess exogenous substrate takes ∼10 min to be complete, whereas the reaction of isolated enzyme in vitro is essentially over in less than 2 min (29)(30)(31)(32). Microsomal preparations of PGHS-1 or -2, which retain the membrane environment but have much lower local phospholipid concentrations than found in intact cells, might be expected to sequester the fatty acid substrate, and indeed the apparent Km value of microsomal COX-1 was twice that found after the membranes were disrupted with detergent (Fig.…”

Section: In Vivo Implications Of Cox Inhibition By Pcmentioning

confidence: 99%

Phospholipid actions on PGHS-1 and -2 cyclooxygenase kinetics

Doyen

Yucer

Lichtenberger

et al. 2008

Prostaglandins & Other Lipid Mediators

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Cyclooxygenase (COX) catalysis by prostaglandin H synthase (PGHS) is a key control step for regulation of prostanoid biosynthesis. Both PGHS isoforms are integral membrane proteins and their substrate fatty acids readily partition into membranes, but the impact of phospholipids and lipid membranes on COX catalysis and the actions of COX inhibitors are not well understood. We have characterized the COX kinetics and ibuprofen inhibition of the purified PGHS isoforms in the presence of phosphatidylcholine (PC) with varying acyl chain structure and physical state. PC was found to directly inhibit COX activity, with non-competitive inhibition by PC monomers binding away from the COX active site and competitive inhibition by micellar/bilayer forms of PC due to sequestration of the arachidonate substrate. Competitive inhibition by native membranes was observed in a comparison of COX kinetics in sheep seminal vesicle microsomes before and after solubilization of PGHS-1. PC liposomes significantly increase the inhibitory potency of ibuprofen against both PGHS isoforms without changing the reversible character of ibuprofen action or requiring binding of PGHS to the liposomes. These results suggest a useful conceptual framework for analyzing the complex interactions among the PGHS proteins, substrates, inhibitors and phospholipid.

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“…AA is metabolized to TXA 2 via two enzymatic steps, including prostaglandin (PG) H and TXA 2 synthase. PGH synthase exhibits both COX and peroxidase activities [22,37]. COX is the target of aspirin and related nonsteroidal antiinflammatory drugs [38].…”

Section: Discussionmentioning

confidence: 99%

“…Microsomes prepared from bovine seminal vesicles were used to determine COX activity, as described by Van Der Ouderaa et al [22]. The COX assay was performed at 37 8C by monitoring oxygen con-sumption using an O2 electrode (Yellow Springs Instrument, Yellow Springs, OH).…”

Section: Assay Of Cox Activitymentioning

confidence: 99%