Human cyclooxygenase-1 activity and its responses to COX inhibitors are allosterically regulated by nonsubstrate fatty acids

Abstract

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...

Competition and allostery govern substrate selectivity of cyclooxygenase-2

“…Since that time, researchers have generally agreed that the COX isoforms do not behave as allosteric enzymes with regard to AA. However, recent evidence strongly supports the concept that the enzymes are functional heterodimers and that activity is modulated by binding of nonsubstrate ligands to the allosteric subunit (8)(9)(10). Here, we report that AA and 2-AG, despite similar catalytic efficiencies with COX-2 when measured individually in vitro, differ markedly in their rate of oxygenation in the presence of the other substrate.…”

“…The homodimeric COX enzymes function as heterodimers, with one subunit that contains the required heme cofactor acting as a catalytic site and the second subunit, which lacks heme, acting as an allosteric site (8,9). Evidence for allosteric regulation of COX is seen in the ability of various nonsubstrate fatty acids to modulate the activity and inhibitor sensitivity of the COXs, presumably through interaction with the allosteric site (8).…”

Metabolism and phospholipid assembly of polyunsaturated fatty acids in human bone marrow mesenchymal stromal cells

“…Because the hBMSC level of 22:4n-6 was almost 4-fold compared with the FBS level, the cells apparently had elongated part of the received bioactive 20:4n-6 to 22:4n-6. Due to its inhibitory effects on COX-1 and COX-2 activities, 22:4n-6 is regarded as biologically less active than 20:4n-6 (50,51). In n-3 PUFAs, a similar, but less extensive, elongation of 20:5n-3 was seen, and consequently 22:5n-3, a more potent COX-1 and COX-2 inhibitor than 20:5n-3 (51, 52), amplifying and spreading the signal.…”

Pre-existent Asymmetry in the Human Cyclooxygenase-2 Sequence Homodimer

“…Titration of huPGHS-2 with Heme-Difference absorption spectra of recombinant forms of apo-huPGHS-2 were obtained upon titration with heme, and K d values for heme and heme binding stoichiometries were calculated as described previously (24,28).…”

“…Dilution of the enzyme sample into the assay chamber was such that the concentrations of inhibitor or PA had no effect on COX activity independent of the time-dependent inhibition. to complete inhibition of COX activity (22), whereas with PGHS-2, the oxygenase activity is decreased by about 50%, and PGH 2 and (15R)-HPETE are formed in comparable amounts (Table 4) (23,24,28,(35)(36)(37). Aspirin acetylation can be used as a quantitative marker for E cat of Native/Native huPGHS-2 (24).…”