Substitution of tyrosine for the proximal histidine ligand to the heme of prostaglandin endoperoxide synthase 2: implications for the mechanism of cyclooxygenase activation and catalysis.
Prostaglandin H(2) synthesis by prostaglandin endoperoxide synthase (PGHS) requires the heme-dependent activation of the protein's cyclooxygenase activity. The PGHS heme participates in cyclooxygenase activation by accepting an electron from Tyr385 located in the cyclooxygenase active site. Two mechanisms have been proposed for the oxidation of Tyr385 by the heme iron: (1) ferric enzyme oxidizes a hydroperoxide activator and the incipient peroxyl radical oxidizes Tyr385, or (2) ferric enzyme reduces a hydroperoxide activator and the incipient ferryl-oxo heme oxidizes Tyr385. The participation of ferrous PGHS in cyclooxygenase activation was evaluated by determining the reduction potential of PGHS-2. Under all conditions tested, this potential (<-135 mV) was well below that required for reactions leading to cyclooxygenase activation. Substitution of the proximal heme ligand, His388, with tyrosine was used as a mechanistic probe of cyclooxygenase activation. His388Tyr PGHS-2, expressed in insect cells and purified to homogeneity, retained cyclooxygenase activity but its peroxidase activity was diminished more than 300-fold. Concordant with this poor peroxidase activity, an extensive lag in His388Tyr cyclooxygenase activity was observed. Addition of hydroperoxides resulted in a concentration-dependent decrease in lag time consistent with each peroxide's ability to act as a His388Tyr peroxidase substrate. However, hydroperoxide treatment had no effect on the maximal rate of arachidonate oxygenation. These data imply that the ferryl-oxo intermediates of peroxidase catalysis, but not the Fe(III)/Fe(II) couple of PGHS, are essential for cyclooxygenase activation. In addition, our findings are strongly supportive of a branched-chain mechanism of cyclooxygenase catalysis in which one activation event leads to many cyclooxygenase turnovers.
Goodwin
D C
DC
Departments of Biochemistry and Chemistry, Center in Molecular Toxicology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Rowlinson
S W
SW
Marnett
L J
LJ
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001665:Binding Sites; D051546:Cyclooxygenase 2; D004579:Electron Transport; D004789:Enzyme Activation; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D007501:Iron / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010544:Peroxidases / Q000378:metabolism; D010545:Peroxides / Q000494:pharmacology; D044262:Prostaglandin H2; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000235:genetics; D011463:Prostaglandins H / Q000096:biosynthesis; D013053:Spectrophotometry; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D007527:Isoenzymes; D010545:Peroxides; D011463:Prostaglandins H; D016718:Arachidonic Acid; D044262:Prostaglandin H2; D014443:Tyrosine; D006418:Heme; D006639:Histidine; D007501:Iron; D010544:Peroxidases; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases