COMPARISON OF PEROXIDASE REACTION-MECHANISMS OF PROSTAGLANDIN-H SYNTHASE-1 CONTAINING HEME AND MANGANO PROTOPORPHYRIN-IX

Prostaglandin H synthase (PGHS) is a heme protein that catalyzes both the cyclooxygenase and peroxidase reactions needed to produce prostagl andins G(2) and H-2 from arachidonic acid. Replacement of the heme gro up by mangano protoporphyrin 1X largely preserves the cyclooxygenase a ctivity, but lowers the steady-state peroxidase activity by 25-fold. T hus, mangano protoporphyrin IX serves as a useful tool to evaluate the function of the heme in PGHS. A detailed kinetic analysis of the pero xidase reaction using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), EtOOH, and other peroxides as substrates has been carried out to compa re the characteristics of PGHS reconstituted with mangano protoporphyr in IX (Mn-PGHS) to those of the native heme enzyme (Fe-PGHS). The rate constant describing the reaction of Mn-PGHS with 15-HPETE to form the oxidized, Mn(IV) intermediate with absorption at 420 nm, exhibits sat urable behavior as the 15-HPETE concentration is raised from 10 to 400 mu M. This is most likely due to the presence of a second, earlier in termediate between the resting enzyme and the Mn(IV) species. Measurem ents at high substrate concentrations permitted resolution of the abso rbance spectra of the two oxidized Mn-PGHS intermediates. The spectrum of the initial intermediate, assigned to a Mn(V) species, had a line shape similar to that of the later intermediate, assigned to a Mn(IV) species, suggesting that a porphyrin pi-cation radical is not generate d in the peroxidase reaction of Mn-PGHS. The rate constant estimated f or the formation of the earlier intermediate with 15-HPETE is 1.0 x 10 (6) M(-1) s(-1) (20 degrees C, pH 7.3). A rate constant of 400 +/- 100 s(-1) was estimated for the second step in the reaction. Thus, Mn-PGH S reacts considerably more slowly than Fe-PGHS with 15-HPETE to form t he first high-valent intermediate, but the two enzymes appear to follo w a similar overall reaction mechanism for generation of oxidized inte rmediates. The difference in rate constants explains the observed lowe r steady-state peroxidase activity of Mn-PGHS compared with Fe-PGHS.