Prostaglandin H synthase: spectroscopic studies of the interaction with hydroperoxides and with indomethacin

Abstract

Prostaglandin H synthase has both a heme-dependent peroxidase activity and a cyclooxygenase activity. A current hypothesis considers the cyclooxygenase reaction to be a free radical chain reaction, initiated by an interaction of the synthase peroxidase with hydroperoxides leading to the production of a tyrosyl free radical [Stubbe, J. A. (1989) Annu. Rev. Biochem. 58, 257-285]. We have examined the kinetics of radical formation with both ethyl hydroperoxide (EtOOH) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and have analyzed the effects of indomethacin (a selective cyclooxygenase inhibitor) and tetranitromethane (TNM; a selective agent for nitration of tyrosyl residues) on the synthase. At -14 degrees C both EtOOH and 15-HPETE generated within 5 s a free radical species whose electron paramagnetic resonance spectrum was dominated by a doublet centered at g = 2.005 (splitting of approximately 16 G; overall peak-to-trough width of 35 G) that has been attributed to tyrosyl radical. The doublet subsequently gave way to a singlet with a similar peak-to-trough width; the doublet-to-singlet transition was complete in 20-60 s. The intensity of the doublet/singlet combination peaked at 0.6 spins/heme after 120 s with EtOOH and at about 0.3 spins/heme after 20 s with 15-HPETE; the radical intensity declined slowly with EtOOH but more rapidly with 15-HPETE. Reaction of the indomethacin-synthase complex with EtOOH resulted in a narrower (peak-to-trough width of 24 G) singlet free radical signal, with no evidence of an earlier doublet; the intensity of the singlet peaked at 0.45 spins/heme after about 300 s. Reaction of TNM-treated synthase with EtOOH resulted in a singlet almost identical with that seen for the indomethacin-synthase complex. Reaction of the synthase holoenzyme with TNM at pH 8.0 led to inactivation of both cyclooxygenase and peroxidase activity, with the former being lost rapidly and completely while the latter was lost slowly and to about 50%. Ibuprofen, a competitive cyclooxygenase inhibitor, slowed the rate of inactivation of the cyclooxygenase by about 20-fold. The rate of inactivation of the cyclooxygenase activity in synthase apoenzyme by TNM was also about 20-fold less than that observed with the holoenzyme. Amino acid analyses revealed that TNM-reacted holoenzyme with less than 10% residual activity contained 1.8 nitrotyrosines/subunit; apoenzyme reacted under the same conditions had greater than 80% of the original activity and contained 0.7 nitrotyrosine/subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a cellular denitrase activity that reverses nitration of cyclooxygenase

“…This is in agreement with MS analysis of denitration products from NO 2 -calmodulin where 3-AT formation was not detected (49). With respect to COX-1, studies (6,15,34) have shown that only a small percentage of tyrosine residues (ϳ3 tyrosine residues per COX-1 monomer) are subject to NO x -induced nitration. Although separation and detection of free tyrosine can be achieved by HPLC-ECD, it was difficult to accurately assess whether the 3-NT signal loss was associated with a commensurate formation of new tyrosine residues, owing to A: rat lung homogenate in detergent-free buffer was subjected to differential centrifugation to obtain cellular fractions that are mitochondria rich and mitochondria depleted (as shown in the schematic).…”

Prostaglandin Endoperoxide H Synthases

“…The k 1 value for Compound I formation from EtOOH is a tenth of that for 15-HPETE, and the k 1 for H 2 O 2 is less than a hundredth of that for 15-HPETE (74,75). This suggests that it is the nature of the first few atoms neighboring the hydroperoxide group that is the most important determinant of the rate of Compound I formation.…”

Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase.

“…Indomethacin inhibits COX-1 activity by binding to its active site and subsequently rendering it inactive (25), possibly by producing a conformational change in an essential protein radical (26). Although the mechanisms by which aspirin and indomethacin inhibit COX are different, the relative potencies of these drugs were similar in all of the assays used in this study and in those using microsomal preparations from COS-1 transfected cells for assay of COX-1 and COX-2 activity (19).…”