Peroxidase self-inactivation in prostaglandin H synthase-1 pretreated with cyclooxygenase inhibitors or substituted with mangano protoporphyrin IX.
Self-inactivation imposes an upper limit on bioactive prostanoid synthesis by prostaglandin H synthase (PGHS). Inactivation of PGHS peroxidase activity has been found to begin with Intermediate II, which contains a tyrosyl radical. The structure of this radical is altered by cyclooxygenase inhibitors, such as indomethacin and flurbiprofen, and by replacement of heme by manganese protoporphyrin IX (forming MnPGHS-1). Peroxidase self-inactivation in inhibitor-treated PGHS-1 and MnPGHS-1 was characterized by stopped-flow spectroscopic techniques and by chromatographic and mass spectrometric analysis of the metalloporphyrin. The rate of peroxidase inactivation was about 0.3 s(-)1 in inhibitor-treated PGHS-1 and much slower in MnPGHS-1 (0.05 s(-)1); as with PGHS-1 itself, the peroxidase inactivation rates were independent of peroxide concentration and structure, consistent with an inactivation process beginning with Intermediate II. The changes in metalloporphyrin absorbance spectra during inactivation of inhibitor-treated PGHS-1 were similar to those observed with PGHS-1 but were rather distinct in MnPGHS-1; the kinetics of the spectral transition from Intermediate II to the next species were comparable to the inactivation kinetics in each case. In contrast to the situation with PGHS-1 itself, significant amounts of heme degradation occurred during inactivation of inhibitor-treated PGHS-1, producing iron chlorin and heme-protein adduct species. Structural perturbations at the peroxidase site (MnPGHS-1) or at the cyclooxygenase site (inhibitor-treated PGHS-1) thus can influence markedly the kinetics and the chemistry of PGHS-1 peroxidase inactivation.
Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
G
G
Wu
J L
JL
Vuletich
R J
RJ
Kulmacz
Y
Y
Osawa
A L
AL
Tsai
D002851:Chromatography, High Pressure Liquid; D051545:Cyclooxygenase 1; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D005480:Flurbiprofen / Q000494:pharmacology; D006418:Heme / Q000737:chemistry; D007213:Indomethacin / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010544:Peroxidases / Q000037:antagonists & inhibitors*; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011524:Protoporphyrins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D011524:Protoporphyrins; C038654:manganese protoporphyrin; D006418:Heme; D005480:Flurbiprofen; D010544:Peroxidases; D051545:Cyclooxygenase 1; D011451:Prostaglandin-Endoperoxide Synthases; D007213:Indomethacin