The Dynamics of Prostaglandin H Synthases

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...

“…2, 3, and 5). An effect of exogenous peroxide on cyclooxygenase cooperativity is difficult to reconcile with the proposed allosteric mechanism (6,20).…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Chen

Pawelek

Kulmacz

1999

“…3); (c) anandamide (arachidonylethanolamide), which lacks a carboxylate group, is a substrate for hPGHS-2 (36); (d) mutation of Tyr-355, which neighbors Arg-120, to a phenylalanine has little effect on the k cat /K m value for arachidonate oxygenation (36); and (e) the hydrophobic tunnel of the active site of PGHS-2 is larger (i.e. more open and accommodating) than that of PGHS-1 (1,12,16,17).…”

Section: Arg-120 In Cox-2mentioning

confidence: 99%

The Role of Arginine 120 of Human Prostaglandin Endoperoxide H Synthase-2 in the Interaction with Fatty Acid Substrates and Inhibitors

Rieke

Mulichak

Garavito

et al. 1999

104

Arg-120 is located near the mouth of the hydrophobic channel that forms the cyclooxygenase active site of prostaglandin endoperoxide H synthases (PGHSs)-1 and -2. Replacement of Arg-120 of ovine PGHS-1 with a glutamine increases the apparent K m of PGHS-1 for arachidonate by 1,000-fold (Bhattacharyya, D. K., Lecomte, M., Rieke, C. J., Garavito, R. M., and Smith, W. L. (1996) J. Biol. Chem. 271, 2179 -2184). This and other evidence indicate that the guanido group of Arg-120 forms an ionic bond with the carboxylate group of arachidonate and that this interaction is an important contributor to the overall strength of arachidonate binding to PGHS-1. In contrast, we report here that R120Q human PGHS-2 (hPGHS-2) and native hPGHS-2 have very similar kinetic properties, but R120L hPGHS-2 catalyzes the oxygenation of arachidonate inefficiently. Our data indicate that the guanido group of Arg-120 of hPGHS-2 interacts with arachidonate through a hydrogen bond rather than an ionic bond and that this interaction is much less important for arachidonate binding to PGHS-2 than to PGHS-1. The K m values of PGHS-1 and -2 for arachidonate are the same, and all but one of the core residues of the active sites of the two isozymes are identical. Thus, the results of our studies of Arg-120 of PGHS-1 and -2 imply that interactions involved in the binding of arachidonate to PGHS-1 and -2 are quite different and that residues within the hydrophobic cyclooxygenase channel must contribute more significantly to arachidonate binding to PGHS-2 than to PGHS-1. As observed previously with R120Q PGHS-1, flurbiprofen was an ineffective inhibitor of R120Q hPGHS-2. PGHS-2-specific inhibitors including NS398, DuP-697, and SC58125 had IC 50 values for the R120Q mutant that were up to 1,000-fold less than those observed for native hPGHS-2; thus, the positively charged guanido group of Arg-120 interferes with the binding of these compounds. NS398 did not cause time-dependent inhibition of R120Q hPGHS-2, whereas DuP-697 and SC58125 were time-dependent inhibitors. Thus, Arg-120 is important for the time-dependent inhibition of hPGHS-2 by NS398 but not by DuP-697 or SC58125.Prostaglandin endoperoxide H synthases (PGHSs) 1 catalyze the committed step in the formation of prostanoids, the conversion of arachidonic acid to prostaglandin (PG) H 2 (1). There are two PGHS isozymes. PGHS-1 is expressed constitutively in most tissues, and PGH 2 formed via PGHS-1 is involved in modulating and coordinating intercellular, intratissue responses to fluctuations in the levels of circulating hormones. PGHS-2 is expressed inducibly in response to cytokines (2-5), growth factors (6 -8), v-src (9), and tumor promoters (10), and products formed through the action of this isoform appear to play a role in cell growth and differentiation (11). Both PGHS-1 and PGHS-2 have similar kinetic and structural properties (1). Both isoforms catalyze two distinct reactions: (a) a cyclooxygenase reaction involving the bis-oxygenation of arachidonic acid to yield PGG 2 , and (b) a peroxidas...

“…Following oxygenation, a conformational rearrangement of arachidonate occurs (15) that could disrupt this hydrogen-bonding network leading to release of prostaglandin G 2 . Since time-dependent NSAIDs cannot undergo oxygenation, the unproductive hydrogen bond-stabilized complex they form dissociates only slowly (24). Thus, time-dependent inhibition may be a serendipitous outcome of a catalytic mechanism evolved in PGHS to ensure fidelity of the cyclooxygenase reaction.…”

Section: Discussionmentioning

confidence: 99%

“…The conformational change in PGHS-2 that we observed upon arachidonate binding may be a mechanism that signals when the substrate is properly positioned within the cyclooxygenase site. This arachidonate/enzyme conformation is likely stabilized by formation of the same network of hydrogen bonds between amino acid side chains at the mouth of the cyclooxygenase active site that stabilizes binding of time-dependent inhibitors (24). Following oxygenation, a conformational rearrangement of arachidonate occurs (15) that could disrupt this hydrogen-bonding network leading to release of prostaglandin G 2 .…”

Section: Discussionmentioning

confidence: 99%

Arachidonic Acid and Nonsteroidal Anti-inflammatory Drugs Induce Conformational Changes in the Human Prostaglandin Endoperoxide H2 Synthase-2 (Cyclooxygenase-2)

Smith

McCracken

Shin

et al. 2000

By using the technique of site-directed spin labeling combined with EPR spectroscopy, we have observed that binding of arachidonic acid and nonsteroidal antiinflammatory drugs induces conformational changes in the human prostaglandin endoperoxide H 2 synthase enzyme (PGHS-2). Line shape broadening resulting from spin-spin coupling of nitroxide pairs introduced into the membrane-binding helices of PGHS-2 was used to calculate the inter-helical distances and changes in these distances that occur in response to binding various ligands. The inter-residue distances determined for the PGHS-2 holoenzyme using EPR were 1-7.9 Å shorter than those of the crystal structure of the PGHS-2 holoenzyme. However, inter-helical distances calculated and determined by EPR for PGHS-2 complexed with arachidonic acid, flurbiprofen, and SC-58125 were in close agreement with those obtained from the cognate crystal structures. These results indicate that the structure of the solubilized PGHS-2 holoenzyme measured in solution differs from the crystal structure of PGHS-2 holoenzyme obtained by x-ray analysis. Furthermore, binding of ligands induces a conformational change in the holo-PGHS-2, converting it to a structure similar to those obtained by x-ray analysis. Proteolysis protection assays had previously provided circumstantial evidence that binding of heme and non-steroidal anti-inflammatory drugs alters the conformation of PGHS, but the present experiments are the first to directly measure such changes. The finding that arachidonate can also induce a conformational change in PGHS-2 was unexpected, and the magnitude of changes suggests this structural flexibility may be integral to the cyclooxygenase catalytic mechanism.Two prostaglandin endoperoxide H 2 synthase (PGHS) 1 isozymes, PGHS-1 and -2 (also known as cyclooxygenase-1 and -2, or COX-1 and COX-2), produce the intermediate PGH 2 from which prostaglandins, prostacyclin, and thromboxanes are derived. These two enzymes are both pharmacological targets of traditional nonsteroidal anti-inflammatory drugs (NSAIDs) (1). Recently, two new drugs have been developed, Celebrex and Vioxx, that selectively inhibit PGHS-2. These "COX-2 inhibitors" have an improved gastrointestinal safety profile, presumably because they do not interfere with prostaglandin production by PGHS-1 (2). Preferential inhibition by COX-2 inhibitors results in part because these drugs are bulky and fit more readily in the larger active site of COX-2 (2, 3). However, the hallmark of COX-2 inhibitors is their differential mechanism of inhibition for PGHS-2. These drugs are time-dependent inhibitors of the PGHS-2, forming tight binding and slowly reversible complexes with this isozyme but are simple competitive reversible inhibitors of the PGHS-1 isozyme.Formation of the NSAID-enzyme complexes has been proposed to result in conformational changes in PGHS (4). This hypothesis is supported by studies that show that time-dependent inhibitors protect from proteolytic cleavage at Arg-277, 2 an amino acid located on a peptide...