Analysis of Hydroperoxide-Induced Tyrosyl Radicals and Lipoxygenase Activity in Aspirin-Treated Human Prostaglandin H Synthase-2

Abstract

A hydroperoxide-induced tyrosyl radical has been proposed as a key cyclooxygenase intermediate for the "basal" isoform of prostaglandin H synthase (PGHS-1). In the present study with the "inducible" isoform (PGHS-2), hydroperoxide was also found to generate a radical in high yield, a wide singlet at g = 2.0058 (29 G peak to trough). Reaction of PGHS-2 with a tyrosine-modifying reagent, tetranitromethane (TNM), resulted in cyclooxygenase inactivation and a much narrower radical EPR signal (22 G peak to trough). Addition of a cyclooxygenase inhibitor, nimesulide, similarly resulted in a narrow PGHS-2 radical. In PGHS-1, cyclooxygenase inhibition by tyrosine nitration with TNM or by active site ligands leads to generation of a narrow EPR instead of a wide EPR, with both signals originating from authentic tyrosyl radicals, indicating that the hydroperoxide-induced radicals in PGHS-2 are also tyrosyl radicals. Treatment of PGHS-2 with aspirin (acetyl salicylic acid, ASA) was previously shown to result in acetylation of a specific serine residue, cyclooxygenase inhibition, and increased lipoxygenase activity. Acetylation of PGHS-1 by ASA, in contrast, inhibited both lipoxygenase and cyclooxygenase activity. We now have found the ASA-treated PGHS-2 radical to be indistinguishable from that in control PGHS-2. Addition of nimesulide to ASA-treated PGHS-2 inhibited the lipoxygenase and resulted in a narrow radical EPR like that seen in PGHS-2 treated with TNM or nimesulide alone. Retention of PGHS-2 oxygenase activity was thus associated with retention of the native radical, and loss of activity was associated with alteration of the radical. Both native and ASA-treated PGHS-2 produced only the R stereoisomer of 11- and 15-HETE, demonstrating that the lipoxygenase stereochemistry was not changed by ASA. Native and ASA-treated PGHS-2 had lipoxygenase K(m) values considerably higher than that of the control PGHS-2 cyclooxygenase. Taken together, these results suggest that the same PGHS-2 tyrosyl radical serves as the oxidant for both cyclooxygenase and lipoxygenase catalysis and that acetylation of PGHS-2 by ASA favors arachidonate binding in an altered conformation which results in abstraction of the pro-R hydrogen from C13 and formation of 11(R)- and 15(R)-HETE.

Acetylation of prostaglandin H2 synthases by aspirin is inhibited by redox cycling of the peroxidase

“…This suggests that these reversible inhibitors either inhibit the formation of the Tyr385 radical or markedly accelerate its reprotonation, and implies an interaction of these inhibitors in the region of Tyr 385. The effect of aspirin itself (before deacetylation) on the Tyr385 radical is not known; after acetylation by aspirin has taken place, the acetylated PGHS-1 does not exhibit the peroxide-induced Tyr385 radical, but this radical is generated by peroxide in acetylated PGHS-2, consistent with the ability of acetylated PGHS-2 to abstract a hydrogen from carbon 13 of arachidonic acid [28]. Acetylation of the enzyme by aspirin is almost completely abrogated in the Tyr385Phe mutant [29].…”

Structural Characterization of Arachidonyl Radicals Formed by Aspirin-treated Prostaglandin H Synthase-2

“…In contrast, the peroxide-induced tyrosyl radical in ASA-PGHS-2 retains the EPR characteristics of the peroxide-induced tyrosyl radical in native PGHS-2, a 29 -30-G wide singlet (25). In the present study, we have examined whether the wide singlet tyrosyl radical of ASA-PGHS-2 is capable of AA oxidation to support the 11-and 15-lipoxygenase activity.…”

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

“…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).…”