Peroxide-induced radical formation at TYR385 and TYR504 in human PGHS-1.
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 change 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 mutants 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) value for arachidonate of hPGHS-1 were not affected by the Y504F mutation, but the peroxidase specific activity and the K(M) value 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 form a tyrosyl radical. These results indicate that peroxide-induced 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 allowed radical formation 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.
Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
Corina E
CE
Rogge
Wen
W
Liu
Richard J
RJ
Kulmacz
Ah-Lim
AL
Tsai
D016718:Arachidonic Acid / Q000378:metabolism; D002384:Catalysis; D051545:Cyclooxygenase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D014443:Tyrosine / Q000378:metabolism*
D005609:Free Radicals; D016718:Arachidonic Acid; D014443:Tyrosine; D006861:Hydrogen Peroxide; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human