Attachment of substrate metabolite to prostaglandin H synthase upon reaction with arachidonic acid

Prostaglandin H synthase was incubated with 14C]arachidonate and then analyzed by polyacrylamide gel electrophoresis under denaturing conditions and by high pressure liquid chromatography. A maximum of 1 mol of arachidonate metabolite was found to become attached per mol of synthase subunit in a time-dependent process that was much slower than the rate of self-catalyzed inactivation of the cyclooxygenase activity. Incubation of a mixture of the synthase and ovalbumin with 14C]arachidonate resulted in a selective attachment of radiolabel to the synthase. These results suggest the presence of a single site on the synthase that is susceptible to reaction with an arachidonate metabolite.