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.
Rogge
Corina E
CE
Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
Liu
Wen
W
Kulmacz
Richard J
RJ
Tsai
Ah-Lim
AL
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