title: Cyclooxygenase inactivation kinetics during reaction of prostaglandin H synthase-1 with peroxide.
abstract: The peroxidase and cyclooxygenase activities of prostaglandin H synthase-1 (PGHS-1) both become irreversibly inactivated during reaction with peroxide. Sequential stopped-flow absorbance measurements with a chromogenic peroxidase cosubstrate previously were used to evaluate the kinetics of peroxidase inactivation during reaction of PGHS-1 with peroxide [Wu, G., et al. (1999) J. Biol. Chem. 274, 9231-7]. This approach has now been adapted to use a chromogenic cyclooxygenase substrate to analyze the detailed kinetics of cyclooxygenase inactivation during reaction of PGHS-1 with several hydroperoxides. In the absence of added reducing cosubstrates, which maximizes the levels of oxidized enzyme intermediates expected to lead to inactivation, cyclooxygenase activity was lost as fast as, or somewhat faster than, peroxidase activity. Cyclooxygenase inactivation kinetics appeared to be sensitive to the structure of the peroxide used. The addition of reducing cosubstrate during reaction of PGHS-1 with peroxide protected the peroxidase activity to a much greater degree than the cyclooxygenase activity. The results suggest a new concept of PGHS inactivation: that distinct damage can occur at the two active sites during side reactions of Intermediate II, which forms during reaction of PGHS with peroxide and which contains two oxidants, a ferryl heme in the peroxidase site, and a tyrosyl free radical in the cyclooxygenase site.
authors: Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
authors: Gang
authors: G
authors: Wu
authors: Richard J
authors: RJ
authors: Kulmacz
authors: Ah-Lim
authors: AL
authors: Tsai
mesh_terms: D000818:Animals; D001665:Binding Sites; D051545:Cyclooxygenase 1; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006139:Guaiacol / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry; D007700:Kinetics; D008297:Male; D009195:Peroxidase / Q000037:antagonists & inhibitors; D010545:Peroxides / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000378:metabolism*; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep; D013053:Spectrophotometry / Q000379:methods
chemical_list: D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D010545:Peroxides; D006139:Guaiacol; D009195:Peroxidase; D051545:Cyclooxygenase 1; D011451:Prostaglandin-Endoperoxide Synthases