The Aspirin and Heme-binding Sites of Ovine and Murine Prostaglandin Endoperoxide Synthases*

of Ser-530 of

In related studies, a cDNA for mouse PGG/H synthase was cloned and sequenced.
A sequence of 35 residues with Ser-530 at the midpoint was identical in the two proteins.
Thus, Ser-530 does lie in a highly conserved region, probably involved in cyclooxygenase catalysis.
Sequence comparisons of mouse and sheep PGG/H synthase also provided information about the hemebinding site of the enzyme. The sheep HYPR sequence (residues 274-277), which had been proposed to form a portion of the distal heme-binding site, is not conserved in the mouse PGG/H synthase, suggesting that this region is not the distal heme-binding site. One sequence, TIWLREHNRV (residues 303-312 of the sheep enzyme), is very closely related to the sequence TLW(L)LREHNRL common to thyroid peroxidase and myeloperoxidase.
The histidine in this latter sequence is the putative axial heme ligand of these peroxidases. We suggest that the histidine (His-309) of sheep PGG/ H synthase sequence is the axial heme ligand of this enzyme.
* This work was supported in part by United States Public Health Service Grants,DK22042, DK42509, and GM40713 and by a Grantin-aid from the American Heart Association of Michigan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Prostaglandin endoperoxide (PGG/H)' synthase (E.C. 1.14.99.1) catalyzes the conversion of arachidonate to prostaglandin Hz (PGH2), the immediate precursor of PGE2, PGFz,, PGD*, prostacyclin, and thromboxane A*. PGG/H synthase has two different enzymatic activities (l-3): (a) a bis oxygenase (cyclooxygenase) which mediates the formation of PGGz from two molecules of oxygen and a molecule of arachidonate, and (b) a hydroperoxidase which catalyzes a net two electron reduction of the 15-hydroperoxyl group of PGG, yielding PGH2. PGG/H synthase is a membrane-associated protein and can only be removed from membrane preparations with detergents (1,4, 5). The subunit molecular weight, of the sheep PGG/H synthase is about 65,000 as calculated from its primary amino acid sequence (6-8); in addition, the molecule contains an average of three asparagine-linked, mannose-containing oligosaccharides (1). The enzyme from sheep vesicular gland is the one which has been studied most extensively. It exists as a homodimer in detergent solution (1,9,10 determine the rate of inactivation of PGG/H synthase by aspirin, enzyme samples (-5 mg of microsomal protein/ml) were incubated plus and minus 0.1 mM aspirin in 0.1 M Tris-Cl, pH 8.0, at 37 "C, and aliquots were removed at 0, 10,20,30,40,60, and 90 min to measure cyclooxygenase activity as described above; t,,, values were determined from plots of the logarithm of activity uersus time. Less than 10% of the cyclooxygenase activity was lost in the absence of aspirin during a 90-min incubation at 37 "C. Immunoprecipitation of PGG/H Synthase-Suspensions (10% w/ v) of IgG(day-l)-Staphylococcus aureus (control) and IgG(cyo-3)-S. aureuS (anti-PGG/H synthase) complexes in 0.1 M Tris-Cl, pH 7.4, containing 0.1% Tween 20, were prepared as described previously (11). Microsomes prepared from transfected cos-1 cells were solubilized by adding one volume of 0.1 M Tris-Cl, pH 8.0, containing 2% Tween 20 to the microsomal suspension. Samples of solubilized microsomes (50-100 ~1; 25-50 units of cyclooxygenase) were incubated for 2 min at 24 "C with 5-50-~1 aliquots of 10% suspensions of IgG(duy-l)-or IgG(cyo-3)-S. aureus complexes and then centrifuged for 2 min at 500 x g. Aliquots of the resulting supernatants were assayed for cyclooxygenase and hydroperoxidase activities as described above.

Western Transfer
Blotting-Solubilized microsomal membranes from sham-transfected cos-1 cells and cos-1 cells transfected with pCD-PGHS,, or pCD-PGHS,,-A530 were resolved by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose filters (0.45 pM) essentially as described by Towbin et al. (36). Filters were blocked in Tris-buffered saline (TBS, 0.1 M Tris-Cl, pH 7.4, containing 0.15 M NaCl) containing 3% bovine serum albumin and then incubated overnight with a 1:50 dilution of monospecific rabbit anti-PGG/H synthase serum (37) or nonimmune rabbit serum. The filters were washed six times with 0.1% Tween 20 in TBS and then incubated with lo-30 &i of '251-Protein A in 3% bovine serum albumin in TBS for 2 h. Finally, the filters were washed four to six times with 0.1% Tween 20 in TBS and twice in TBS, air-dried, and subjected to autoradiography using Kodak XRP-5 x-ray film.

Oligonucleotide-directed
Mutagenesis of Ser-530-Acetylation of the hydroxyl group of Ser-530 of sheep PGG/H synthase by aspirin causes irreversible inactivation of the cyclooxygenase activity of the enzyme (6-8, 21, 29). To determine what role the hydroxyl group of Ser-530 might play in cyclooxygenase catalysis, we used the following approach to replace Ser-530 with Ala-530 and to compare the native and mutant enzymes: (a) starting with an M13mp19 construct containing the coding region of sheep PGG/H synthase (6), the codon for Ser-530 (nucleotides 1687-1689 (6)) was altered to the codon for alanine using the method of Kunkel et al. (29); (b) the coding regions of the native (Ser-530) and mutant (Ala-530) enzymes were removed from the two M13mp19 constructs and used to prepare Okayama/Berg pCD expression vectors (31, 32); (c) the pCD vectors (designated pCD-PGHS,, and pCD-PGHS,,-A530) were used to transfect host cos-1 cells; and finally, (d) the cyclooxygenase and hydroperoxidase activities of the native and mutant PGG/H synthases expressed by the transfected cell populations were characterized.
Shown in Fig. 2 is an autoradiogram of a sequencing gel comparing the nucleotide sequences of the native and mutant PGG/H synthase in the region coding for amino acid residue number 530 (nucleotides 1687-1689). These sequences are 5'-TCC and 5'-GCC for the native (Ser-530) and mutant (Ala-530) cDNAs, respectively.
Cultures of cos-1 cells were transfected with each of the expression vectors (pCD-PGHS,, and pCD-PGHS,,-A530), and 48 h after transfection, microsomal membranes were prepared from each group of transfected cells. As shown by Western transfer blotting (Fig. 3), both groups of cells con- tained proteins of the same size (Mr = 70,000) which interacted with a rabbit anti-PGG/H synthase antiserum. This serum was shown previously to be monospecific for PGG/H synthase (37). As judged by the labeling intensity, similar concentrations of native and mutant PGG/H synthases were present in microsomal membranes prepared from the transfected cells, while little or no immunoreactivity was apparent in membranes from sham-transfected cells (Fig. 3). No radiolabeling of any proteins was seen in a control blot treated with nonimmune rabbit serum in place of anti-PGG/H synthase serum. Table I compares the cyclooxygenase and hydroperoxidase activities of membranes from sham-transfectedcos-1 cells and cos-1 cells transfected with expression vectors for either the native (pCD-PGHS,,) or mutant (pCD-PGHS,,-A530) PGG/ H synthases. Consistent with results obtained previously, we found essentially no PGG/H synthase activity in sham-transfected cells, but comparatively high levels of both cyclooxygenase and hydroperoxidase activities in cells transfected with pCD-PGHS,, which contains the cDNA coding for the native enzyme (32). The striking new result was that the mutant enzyme exhibited a cyclooxygenase specific activity quite similar to that found for the native enzyme.* Further comparisons of the cyclooxygenase activities of the native and mutant enzymes indicated that these two activities have quite similar kinetic constants. Both enzymes had essentially identical K,,, values for arachidonate; furthermore, the ID50 values for in-* In six separate transfections using different cos-I cell preparations and different preparations of pCD vectors, the levels of cyclooxygenase activity were consistently lo-30% lower for the cells transfected with the mutant as opposed to the native cDNA; furthermore, the ratio of cyclooxygenase to hydroperoxidase activity was lo-30% higher for the native uersus the mutant enzyme (e.g. Table I). We also noted that immunoprecipitation of identical amounts of cyclooxygenase activity with the monoclonal antibody IgG(cyo-3) S. aureus complex required roughly 1.5 times more antibody complex in the case of the mutant enzyme. Assuming that this monoclonal antibody is equally reactive with both the native and mutant PGG/H synthases, these results indicate that the Ala-530 mutant has a lo-30% lower turnover number than the native Ser-530 enzyme. Other kinetic properties of the two enzymes are the same (Table I) Membranes were prepared from transfected and sham-transfected cos-1 cells and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Samples were transferred to nitrocellulose and incubated sequentially with rabbit anti-PGG/H synthase serum and '9-labeled Protein A. The samples were dried and subjected to autoradiography as detailed in the text. Lane A, sheep PGG/H synthase (0.25 rg) purified by immunoaffinity chromatography (6); lone B, microsomes from cos-1 cells transfected with pCD-PGHS,, (30 pg of protein); lone C, microsomes from cos-1 cells transfected with pCD-PGHS,,-A530 (30 pg of protein); and he D, microsomes from sham-transfected cos-1 cells (30 rg of protein). stantaneous, reversible inhibition of the cyclooxygenase activities by the nonsteroidal anti-inflammatory agents, aspirin, flufenamat,e, and flurbiprofen were the same for native and mutant enzymes (Table I).
Because the Ala-530 mutant lacks an hydroxyl group at position 530, it cannot be acetylated by aspirin. Accordingly, the mutant enzyme was found to differ from the native enzyme in being refractory to time-dependent, irreversible inactivation by aspirin (Table I). This finding establishes that it is acetylation of Ser-530 which is responsible for irreversible inactivation of native cyclooxygenase by aspirin. The results in comparing the catalytic properties of native and mutant PGG/H synthases, and the sensitivities of these two enzymes to aspirin inactivation indicate that the hydroxyl group of Ser-530 is not required for substrate binding or cyclooxygenase catalysis; the results further suggest that introducing a bulky group, such as an acetyl group, at position 530 interferes with the cyclooxygenase reaction, perhaps by interfering with arachidonate binding. One would anticipate that introducing a bulky, uncharged amino acid such as asparagine or glutamine at position 530 would also prevent the cyclooxygenase reaction from occurring; preliminary results suggest that this is the case (38).
Although the Ala-530 mutant is insensitive to inactivation by aspirin, both the Ala-530 mutant and the native enzyme underwent time-dependent, irreversible inactivation when incubated with flurbiprofen (data not shown). Thus, irreversible inactivation of PGG/H synthase by flurbiprofen does not require the hydroxyl group of Ser-530.
Finally, as shown in Table I, all of the cyclooxygenase activity and approximately 95% of the hydroperoxidase activity solubilized from membranes from transfected cos-1 cells was precipitated by a monoclonal antibody (IgG (~~0-3); (11, 34)) to PGG/H synthase. This result verifies that both the cyclooxygenase and hydroperoxidase activities being measured in these experiments can be attributed to PGG/H synthase.
Structure of a Mouse 3T3 Cell PGG/H Synthase-Although Ser-530 appears to play no obvious role in the cyclooxygenase reaction, we found, after sequencing a mouse PGG/H synthase, that the region surrounding Ser-530 is very highly conserved. Fig. 4 shows the nucleotide sequence and deduced amino acid sequence of a cDNA coding for a mouse PGG/H synthase. The mouse cDNA shown in Fig. 4 was cloned from a XgtlO cDNA library which was prepared from poly(A)+ RNA isolated from Swiss mouse 3T3 cells; the library was screened with a 1.6-kb EcoRI restriction fragment prepared from the sheep PGG/H synthase cDNA (6). The mouse cDNA for PGG/H synthase contains 2757 bases (Fig. 4). An open reading frame of 1806 bases begins at nucleotide 37 and extends to the stop codon TGA, at nucleotides 1843-1845. There is 81% nucleotide sequence identity between the mouse cDNA ( Fig. 4) and the sheep cDNA for PGG/H synthase (6-8) in the sequences containing the protein coding regions (nucleotides l-2000); there is 50% identity between the sequences in the region (nucleotides 2000-2757) encompassing the 3'-noncoding region.
The mouse cDNA was found to hybridize with a single species (3 kb) of cytoplasmic RNA from mouse 3T3 cells (data not shown). This RNA species is somewhat larger than the mouse cDNA and larger than the cytoplasmic RNA (2.8 kb) for sheep vesicular gland PGG/H synthase (6). We suspect the difference in the size of our cDNA and the observed mouse message is due to an incomplete 5'-noncoding region in our cDNA. Our reasoning is as follows. Although the 3'-noncoding region of the mouse cDNA lacks the poly(A) tail, it is 87% identical over the last 33 nucleotides with the sheep sequence, including the polyadenylation signal AATAAA; we infer from this observation that the poly(A) sequence of the mouse cDNA would begin within a few bases of the 3' end of the mouse cDNA we have cloned; thus, our mouse cDNA is probably lacking a complete 5'-untranslated sequence.
The deduced amino acid sequence of the mouse PGG/H synthase contains 602 amino acids (Fig. 4). Fig. 5 compares the primary amino acid sequences of the mouse and sheep enzyme. There is 88% sequence identity; of the amino acids which are not identical, only 11 (~2%) result from nonconservative codon changes; three of these differences result from insertions and changes in the putative signal peptide and five others are found in the 12 amino acids at the C terminus. Notable similarities between the sheep and mouse PGG/H synthase sequences include (a) four potential sites of Nglycosylation (asparagines 68 (70)  sheep ::.,:::::::::::::::.___.__..._._..::::::::::::::: Identical residues are marked with a colon (:); residues involving a one-letter change in the codon (i.e. conservative substitutions) are marked with a period (.); nonconservative changes involving two-or three-letter changes in the codon are unmarked. Alignments were performed using the FASTP sequence comparison program (45). serine in this region suggest that this area of the protein may be especially important in cyclooxygenase catalysis. A notable difference between the sheep and the mouse PGG/H synthase sequences occurs in a region which includes amino acid residues 270-280 (272-282). The sheep enzyme is selectively cleaved by trypsin at Arg-277; however, there is not an homologous arginine in the mouse protein (although it is conceivable that the mouse enzyme could be cleaved at a nonhomologous Arg (276)). The sheep sequence HYPR (amino acids 274-277) postulated to form part of the distal hemebinding site (39) of PGG/H synthase is not present in the mouse enzyme; the sequence of the corresponding region of the mouse is RYPP (amino acids 276-279). Since heme binding to PGG/H synthase appears to involve imidazole at both the distal and proximal sites (17) and PGG/H synthase sequences. However, the overall amino acid sequence identities between the two PGG/H synthases and the thyroid and myeloperoxidases are less than 20%. The histidine residue present in the TLW(L)LREHNRL sequences of thyroid peroxidase and myeloperoxidase has been proposed as a potential site for axial heme binding (43). By analogy, we suggest that the histidine residue in the TIWLREHNRV sequence of mouse and sheep PGG/H synthases (His-309 of the sheep; His-311 of the mouse) is the axial heme ligand of this enzyme.
The location of the distal histidine residue is not obvious. In plant and mammalian peroxidases, the distal histidine is present in a sequence, RXXXH (43). There is no histidine present in PGG/H synthase in a sequence of this type. There is, however, one sequence, KALGH (residues 222-226 of the sheep; 224-228 of the mouse), which is found in sheep and mouse PGG/H synthases in a region encompassing 44 amino acids (residues 198-242 of the sheep; 200-244 of the mouse) in which the sequences of sheep and mouse PGG/H synthases are identical (Fig. 5). The KALGH sequence has a basic amino acid (i.e. lysine) 4 residues removed from a histidine, and thus, is similar to the RXXXH of other peroxidases. It is conceivable that His-226 of the sheep (His-228 of the mouse) is the distal histidine. Importantly, there are only 13 histidines (including His-309) (311) and His-226 (228), the putative proximal and distal histidines, respectively) which are con-