Elizabeth D. Thuresson

The Membrane Binding Domains of Prostaglandin Endoperoxide H Synthases 1 and 2 PEPTIDE MAPPING AND MUTATIONAL ANALYSIS

Prostaglandin endoperoxide H synthases 1 and 2 (PGHS-1 and -2) are the major targets of nonsteroidal anti-inflammatory drugs. Both isozymes are integral membrane proteins but lack transmembrane domains. X-ray crystallographic studies have led to the hypothesis that PGHS-1 and -2 associate with only one face of the membrane bilayer through a novel, monotopic membrane binding domain (MBD) that is comprised of four short, consecutive, amphipathic α-helices (helices A–D) that include residues 74–122 in ovine PGHS-1 (oPGHS-1) and residues 59–108 in human PGHS-2 (hPGHS-2). Previous biochemical studies from our laboratory showed that the MBD of oPGHS-1 lies somewhere between amino acids 25 and 166. In studies reported here, membrane-associated forms of oPGHS-1 and hPGHS-2 were labeled using the hydrophobic, photoactivable reagent 3-trifluoro-3-(m-[125I]iodophenyl)diazirine, isolated, and cleaved with AspN and/or GluC, and the photolabeled peptides were sequenced. The results establish that the MBDs of oPGHS-1 and hPGHS-2 reside within residues 74–140 and 59–111, respectively, and thus provide direct provide biochemical support for the hypothesis that PGHS-1 and -2 do associate with membranes through a monotopic MBD. We also prepared HelA, HelB, and HelC mutants of oPGHS-1, in which, for each helix, three or four hydrophobic residues expected to protrude into the membrane were replaced with small, neutral residues. When expressed in COS-1 cells, HelA and HelC mutants exhibited little or no catalytic activity and were present, at least in part, as misfolded aggregates. The HelB mutant retained about 20% of the cyclooxygenase activity of native oPGHS-1 and partitioned in subcellular fractions like native oPGHS-1; however, the HelB mutant exhibited an extra site of N-glycosylation at Asn104. When this glycosylation site was eliminated (HelB/N104Q mutation), the mutant lacked cyclooxygenase activity. Thus, our mutational analyses indicate that the amphipathic character of each helix is important for the assembly and folding of oPGHS-1 to a cyclooxygenase active form.

Pgg 2 11r-Hpete and 15r/S-Hpete are Formed From Different Conformers of Arachidonic Acid in the Prostaglandin Endoperoxide H Synthase-1 Cyclooxygenase Site

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) catalyze the committed step in the formation of prostanoids (prostaglandins, thromboxane A2 (l-6). PGHSs catalyze two separate reactions: a cyclooxygenase reaction in which arachidonate is converted to prostaglandin G2 (PGG2) and a peroxidase reaction in which PGG2undergoes a two-electron reduction to PGH2. The cyclooxygenase reaction begins with a rate-limiting abstraction of the 13-proS hydrogen from arachidonate to yield an arachidonyl radical (7,8). This is followed by sequential oxygen additions at C-11 and C-15 to yield the prostaglandin endoperoxide PGG2. PGHSs exhibit some lipoxygenase activity producing small amounts of 11-hydroperoxy-5Z,8Z,12E,14Z-eicosatetraenoic acid (11-HPETE) and 15-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HPETE) from arachidonic acid (9, 10). Aspirin-acetylated PGHS-2, which has no cyclooxygenase activity, synthesizes 15R-HPETE (10,11). Studies comparing native and aspirinacetylated PGHS-2 have raised the possibility that arachidonate can bind in distinct orientations in the PGHS-2 active site to produce either PGG2, 1 l R-HPETE or 15RHPETE (10). Here we develop the concept that arachidonate can be bound in the cyclooxygenase active site of ovine (o)PGHS-1 in at least three different, catalytically competent arrangements that lead to PGG2, 11R-HPETE, and 15R/S-HPETE, respectively, and that these three arrangements of arachidonate occur subsequent to its entry into the cyclooxygenase active site.

Fatty-Acid Substrate Interactions with Cyclo-oxygenases

Prostaglandin endoperoxide H synthases 1 and -2 (PGHS-1 and -2) convert arachidonic acid and O2 (along with two reducing equivalents) to PGH2 — the committing step in the formation of prostanoids (Smith and DeWitt 1996; Smith et al. 1996). PGHS-1 is often referred to as the constitutive enzyme, whereas PGHS-2 is known as the inducible isoform. They differ from one another mainly with respect to their temporal patterns of expression. The reason for the existence of the two PGHS isozymes is still unknown. One possibility is that PGHS-2 is induced and then functions at relatively low fatty-acid substrate and hydroperoxide-activator concentrations to generate prostanoid products during early stages of cell replication or differentiation, whereas PGHS-1 forms products that are involved in “housekeeping” functions when circulating hormones act on cells acutely to cause the release of higher concentrations of arachidonate (Capdevila et al. 1995; Kulmacz and Wang 1995; Kulmacz 1998; So et al. 1998).

Fatty acid binding to cyclooxygenases

Abstract Prostaglandin endoperoxide H synthase-1 and -2 (PGHS-1 and -2) convert arachidonic acid (AA) to prostaglandin H 2 (PGH 2 ) in the committed step in prostaglandin biosynthesis. Although the cyclooxygenase activity favors AA as the substrate, both isoforms will oxygenate a variety of 18–22 carbon fatty acids with reduced efficiencies. In this review, we discuss how the fatty acid substrates AA and dihomo-γ-linolenic acid (DHLA; 20:3 ω−6) are bound in the cyclooxygenase active site of ovine (o) PGHS-1. Based on the conformations of the fatty acids within the active site, we describe the key roles that Val349 and Ser530 play as determinants of fatty acid substrate specificity for oPGHS-1.

Substrate Interactions in the Cyclooxygenase-1 Active Site

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) catalyze the conversion of arachidonic acid, two molecules of O2 and two electrons to PGH2. This is the committed step in the formation of prostaglandins and thromboxane A2 [1]. Crystallographic studies of enzyme inhibitor complexes have suggested that the cyclooxygenase active sites of PGHSs are hydrophobic channels that protrude into the body of the major globular domain of the enzymes [2]. We have now determined the structure of arachidonic acid (AA) bound within the cyclooxygenase active site of ovine (o)PGHS-1 [3]. AA is bound in an extended L-shaped conformation and makes a total of 49 hydrophobic contacts (i.e. 2.5–4.0 A) and two hydrophilic contacts with the protein involving a total of 19 different residues (Figures 1,2). Although AA can assume some 107 low energy conformations [4], only three of these are catalytically competent [5]. One conformation leads to PGG2, one leads to 11R-HPETE, and a third leads to 15R- plus 15S-HPETE. Previous mutational studies have established the importance of Arg120 in AA binding to PGHS-1 and -2 [6–8], the role of Tyr385 in abstraction of the 13-proS-hydrogen from AA [9,10] and the importance of Ser530 and I1e523 as determinants of inhibitor specificity [11–13]. We have performed mutational analyses of a number of the residues that line the cyclooxygenase channel to determine their functional importance in AA binding and oxygenation. Substitutions of several cyclooxygenase site residues lead to large increases in 11-HPETE or 15-HPETE formation but with small changes in the Km for AA. Our results suggest that individually and collectively the hydrophobic residues function primarily to position AA in a specific conformation that optimizes its conversion to PGG2.