Nitric Oxide Trapping of Tyrosyl Radicals Generated during Prostaglandin Endoperoxide Synthase Turnover

Abstract

Tyrosyl radicals have been detected during turnover of prostaglandin endoperoxide H synthase (PGHS), and they are speculated to participate in cyclooxygenase catalysis. Spectroscopic approaches to elucidate the identity of the radicals have not been definitive, so we have attempted to trap the radical(s) with nitric oxide (NO). NO quenched the EPR signal generated by reaction of purified ram seminal vesicle PGHS with arachidonic acid, suggesting that NO coupled with a tyrosyl radical to form inter alia nitrosocyclohexadienone. Subsequent formation of nitrotyrosine was detected by Western blotting of PGHS incubated with NO and arachidonic acid or organic hydroperoxides using an antibody against nitrotyrosine. Both arachidonic acid and NO were required to form nitrotyrosine, and tyrosine nitration was blocked by the PGHS inhibitor indomethacin. The presence of superoxide dismutase had no effect on nitration, indicating that peroxynitrite was not the nitrating agent. To identify which tyrosines were nitrated, PGHS was digested with trypsin, and the resulting peptides were separated by high pressure liquid chromatography and monitored with a diode array detector. A single peptide was detected that exhibited a spectrum consistent with the presence of nitrotyrosine. Consistent with Western blotting results, both NO and arachidonic acid were required to observe nitration of this peptide, and its formation was blocked by the PGHS inhibitor indomethacin. Peptide sequencing indicated that the modified residue was tyrosine 385, the source of the putative catalytically active tyrosyl radical.Prostaglandin endoperoxide H synthase (PGHS), 1 a bifunctional heme enzyme, catalyzes the first two steps of prostaglandin and thromboxane biosynthesis. Its cyclooxygenase activity catalyzes the incorporation of two molecules of dioxygen into arachidonic acid to form prostaglandin endoperoxide G 2 , a hydroperoxy endoperoxide (Reaction 1). The peroxidase activity of PGHS then catalyzes the twoelectron reduction of prostaglandin G 2 to prostaglandin endoperoxide H 2 , a hydroxy endoperoxide (Reaction 2) (1-3).The mechanism of prostaglandin synthesis by PGHS, particularly the relationship between the two activities of this enzyme, has been the subject of intense investigation. The peroxidase and cyclooxygenase activities of PGHS are separate and distinct (4). The two active sites are located on opposite sides of the protein and are separated by the heme prosthetic group. Nonsteroidal antiinflammatory drugs that bind in the cyclooxygenase active site do not inhibit the peroxidase (3). Despite this spatial separation the peroxidase and cyclooxygenase activities are functionally interconnected. Ligands to the distal heme binding site inhibit both activities (5-7). Scavenging of fatty acid hydroperoxides with glutathione peroxidase inhibits cyclooxygenase activity, and protein or heme modifications that reduce peroxidase activity induce a lag phase in cyclooxygenase activity that can be overcome by addition of hydroperoxide (8,9). It is belie...

Nitric Oxide Synthase/COX Cross-Talk: Nitric Oxide Activates COX-1 But Inhibits COX-2-Derived Prostaglandin Production

“…Thus, these findings indicate that NO induces the nitration of COX-2 in LPS-stimulated macrophages. As has been shown in cell-free systems by Gunther et al (9), it is likely that such nitration inhibits the catalytic activity of the enzyme, as those authors have reported (8,9). Whether COX-2 nitration decreases the stability of the enzyme and accounts for decreased COX-2 expression in NO-treated cells remains to be determined.…”

“…Tyr 385 contributes to enzyme activity; in addition, the tyrosyl radical species is involved in suicide inactivation (7). In cell-free systems, NO has been reported to trap the tyrosyl radical of the COXs, which leads to tyrosine iminoxyl radical formation and inactivation of the enzyme (8,9). The PG-biosynthetic pathway is initiated by activation of phospholipase A 2 (PLA 2 ), which are primarily responsible for agonistinduced arachidonic acid release from membrane phospholipids (10).…”

Spatial Requirements for 15-(R)-Hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic Acid Synthesis within the Cyclooxygenase Active Site of Murine COX-2

“…The carboxylate moiety is anchored to Arg-120 and Tyr-355, and the fatty acid backbone extends upwards toward the top of the COX active site, with the 13-pro-S hydrogen of the substrate positioned close to Tyr-385 (32). The -end of arachidonate extends into the region we have termed the top channel, with carbons 17-20 lying above Ser-530.…”

Nitric Oxide Regulation of Free Radical– and Enzyme-Mediated Lipid and Lipoprotein Oxidation

“…In fact, ONOO Ϫ stimulates PGHS cyclooxygenase activity even in the presence of hydroperoxide-scavenging reaction systems, such as glutathioneϩGPx, 47,48 resulting in the proposal that ONOO Ϫ is a central mediator in tissue PGHS activation mechanisms.…”