Reaction of prostaglandin endoperoxide synthase with cis,cis-eicosa-11,14-dienoic acid

The pre-steady-state phase of the oxygenase reaction of prostaglandin endoperoxide synthase with cis,cis-eicosa-11, 14-dienoic acid has been studied using stopped flow techniques. Because some intermediate forms of prostaglandin endoperoxide synthase are spectrally indistinguishable, the enzyme and substrate transformations were monitored in parallel to simplify the interpretation of the kinetics. Over a wide range of conditions, the formation of the enzyme intermediate II, the form of compound I containing the tyrosyl radical, precedes substrate oxidation. This result supports the occurrence of a unimolecular conversion of compound I into intermediate II. Furthermore, the rate of intermediate II formation was stimulated by increased concentration of dienoic acid, perhaps because of increased occupation of the fatty acid binding site. The importance of the unimolecular formation of intermediate II was confirmed by simulated kinetics of the oxygenase reaction. These results provide evidence that intermediate II is the primary oxidant in the reaction of prostaglandin synthase with the dienoic acid, as it is with arachidonic acid.