Prostaglandin endoperoxide H synthases (PGHSs), also called cyclooxygenases (COXs), convert arachidonic acid (AA) to PGH2. PGHS-1 and PGHS-2 are conformational heterodimers, each composed of an (Eallo) and a catalytic (Ecat) monomer. Previous studies suggested that the binding to Eallo of saturated or monounsaturated fatty acids (FAs) that are not COX substrates differentially regulate PGHS-1 versus PGHS-2. Here, we substantiate and expand this concept to include polyunsaturated FAs known to modulate COX activities. Non-substrate FAs like palmitic acid bind Eallo of PGHSs stimulating human (hu) PGHS-2 but inhibiting huPGHS-1. We find the maximal effects of non-substrate FAs on both huPGHSs occurring at the same physiologically relevant FA/AA ratio of ∼20. This inverse allosteric regulation likely underlies the ability of PGHS-2 to operate at low AA concentrations, when PGHS-1 is effectively latent. Unlike FAs tested previously, we observe that C-22 FAs, including ω-3 fish oil FAs, have higher affinities for Ecat than Eallo subunits of PGHSs. Curiously, C-20 ω-3 eicosapentaenoate preferentially binds Ecat of huPGHS-1 but Eallo of huPGHS-2. PGE2 production decreases 50% when fish oil consumption produces tissue EPA/AA ratios of ≥0.2. However, 50% inhibition of huPGHS-1 itself is only seen with ω-3 FA/AA ratios of ≥5.0. This suggests that fish oil-enriched diets disfavor AA oxygenation by altering the composition of the FA pool in which PGHS-1 functions. The distinctive binding specificities of PGHS subunits permit different combinations of non-esterified FAs, which can be manipulated dietarily, to regulate AA binding to Eallo and/or Ecat thereby controlling COX activities.

This article is available online at http://www.jlr.org formation of 1-, 2-and 3-series prostaglandins from dihomo-␥ -linolenic acid (DHLA), arachidonic acid (AA), and eicosapentaenoic acid (EPA), respectively. PGHSs are commonly called cyclooxygenases (COXs). There are two PGHS isoforms encoded by different genes and known as PGHS-1 and PGHS-2 or COX-1 and COX-2.PGHSs catalyze both COX and peroxidase (POX) reactions that occur at distinct physical sites on the enzymes but sites that are electronically and mechanistically linked. The COX is a bis -oxygenase. During the COX reaction with AA, two O 2 molecules are introduced into the carbon backbone of the substrate, and various rearrangements occur that yield a bicyclic endoperoxide called prostaglandin G 2 (PGG 2 ). The 15-hydroperoxyl group of PGG 2 can be reduced to prostaglandin H 2 by the POX activity. The POX and COX activities of PGHSs require hydroperoxides ( 4, 6 ). Hydroperoxides are immediate substrates for the POX activity of PGHS. Oxidation of the heme group at the POX site during the reduction of peroxides leads to the formation of a tyrosyl radical in the COX site. The tyrosyl radical abstracts a hydrogen from AA in the initial step in COX catalysis. Ovine (ov) PGHS-1 prefers primary and secondary alkyl hydroperoxides to H 2 O 2 or bulky peroxides like t -butyl hydroperoxide ( 7,8 ).PGHSs are targets of COX inhibitors, including nonspecifi c nonsteroidal anti-infl ammatory drugs (nsNSAIDs) that inhibit both isoforms and COX-2 inhibitors that are relatively more specifi c for the COX activity of PGHS-2; the more recently developed COX-2 inhibitors are often called coxibs. Mechanistically, COX inhibitors fall into two broad categories, time-dependent and time-independent/ Abstract Recombinant human prostaglandin endoperoxide H synthase-1 (huPGHS-1) was characterized. huPGHS-1 has a single high-affi nity heme binding site per dimer and exhibits maximal cyclooxygenase (COX) activity with one heme per dimer. Thus, huPGHS-1 functions as a conformational heterodimer having a catalytic monomer (E cat ) with a bound heme and an allosteric monomer (E allo ) lacking heme. The enzyme is modestly inhibited by common FAs including palmitic, stearic, and oleic acids that are not COX substrates. Studies of arachidonic acid (AA) substrate turnover at high enzyme-to-substrate ratios indicate that nonsubstrate FAs bind the COX site of E allo to modulate the properties of E cat . Nonsubstrate FAs slightly inhibit huPGHS-1 but stimulate huPGHS-2, thereby augmenting AA oxygenation by PGHS-2 relative to PGHS-1. Nonsubstrate FAs potentiate the inhibition of huPGHS-1 activity by time-dependent COX inhibitors, including aspirin, all of which bind E cat . Surprisingly, preincubating huPGHS-1 with nonsubstrate FAs in combination with ibuprofen, which by itself is a time-independent inhibitor, causes a short-lived, time-dependent inhibition of huPGHS-1. Thus, in general, having a FA bound to E allo stabilizes time-dependently inhibited conformations of E cat . We speculate that...

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