Peroxide-induced radical formation at TYR385 and TYR504 in human PGHS-1

Abstract

Prostaglandin H synthase isoforms 1 and -2 (PGHS-1 and -2) react with peroxide to form a radical on Tyr385 that initiates the cyclooxygenase catalysis. The tyrosyl radical EPR signals of PGHS-1 and -2 changes over time and are altered by cyclooxygenase inhibitor binding. We characterized the tyrosyl radical dynamics using wild type human PGHS-1 (hPGHS-1) and its Y504F, Y385F and Y385F/Y504F mutats to determine whether the radical EPR signal changes involve Tyr504 radical formation, Tyr385 radical phenyl ring rotation, or both. Reaction of hPGHS-1 with peroxide produced a wide singlet, whereas its Y504F mutant produced only a wide doublet signal, assigned to the Tyr385 radical. The cyclooxygenase specific activity and K M for arachidonate of hPGHS-1 were not affected by the Y504F mutation, but the peroxidase specific activity and the K M for peroxide were increased. The Y385F and Y385F/Y504F mutants retained only a small fraction of the peroxidase activity; the former had a much-reduced yield of peroxide-induced radical and the latter essentially none. After binding of indomethacin, a cyclooxygenase inhibitor, hPGHS-1 produced a narrow singlet but the Y504F mutant did not show much radical. These results indicate that peroxideinduced radicals form on Tyr385 and Tyr504 of hPGHS-1, with radical primarily on Tyr504 in the wild type protein; indomethacin binding prevented radical formation on Tyr385 but still on Tyr504. Thus, hPGHS-1 and -2 have different distributions of peroxide-derived radical between Tyr385 and Tyr504. Y504F mutants in both hPGHS-1 and -2 significantly decreased the cyclooxygenase activation efficiency, indicating that formation of the Tyr504 radical is functionally important for both isoforms.

Asymmetric Acetylation of the Cyclooxygenase-2 Homodimer by Aspirin and Its Effects on the Oxygenation of Arachidonic, Eicosapentaenoic, and Docosahexaenoic Acids

“…Ser530 is not required for inhibition by indomethacin, flurbiprofen, or meclofenamate, which are also maximally effective at one inhibitor molecule per dimer (Kulmacz and Lands, 1985;DeWitt et al, 1990;Yuan et al, 2006Yuan et al, , 2009Prusakiewicz et al, 2009) and mediate an inhibition requiring interaction with Arg120 (Bhattacharyya et al, 1996;Rieke et al, 1999;Rowlinson et al, 2003;Yuan et al, 2006) but not Ser530 (Rowlinson et al, 2003). Inhibition by some nsNSAIDs is associated with decreased formation of the Tyr385 radical involved in catalysis (Rogge et al, 2006(Rogge et al, , 2009. It is not known whether this is the case with diclofenac, but Ser530 does lie across from Tyr385 in the COX active site channel, perhaps with an intervening water molecule (Selinsky et al, 2001).…”

Prostaglandin H synthase: Resolved and unresolved mechanistic issues

“…In a recent similar study on human PGHS-1 (hPGHS-1), the Y504F single mutant produced only a WD tyrosyl radical upon reaction with peroxide (Fig. 12, right panel) [69]. The Y385F/Y504F double mutant of PGHS-1 had no peroxide-induced radical.…”

Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2

“…The EPR lineshape of this NS radical was very similar to that of the NS1a species observed when indomethacin-treated PGHS-1 was reacted with hydroperoxide [20]. NS1a appears to arise from the formation of radical at Tyr504 instead of Tyr385 [18,19]. The mechanism by which AA binding would alter the distribution of tyrosyl radical from Tyr385 to Tyr504 is unclear.…”

“…These WS signals are made up of the original WD and a narrow singlet spectrum (NS1 and NS2 for PGHS-1 and -2, respectively) [14]. Results from EPR studies of peroxide generated radicals in PGHS-1 and -2 with mutated tyrosine residues indicate that the WD→WS spectral transition involves formation of tyrosyl radicals at Tyr385 and Tyr504, with the Tyr385 radical (WD) dominating in the early stages and the Tyr504 (NS) predominating later [17–19]. When PGHS is first complexed with some cyclooxygenase inhibitors, reaction with peroxide generates narrow singlet tyrosyl radicals, NS1a for PGHS-1 and NS2 for PGHS-2 [13–15,20].…”