Stoichiometry of the interaction of prostaglandin H synthase with substrates.

Prostaglandin H synthase (PGHS) catalyzes both peroxidase and cyclooxygenase reactions. Resolution of several current issues regarding the PGHS catalytic mechanism hinges on the stoichiometry of the reaction of PGHS with hydroperoxide, fatty acid, and oxygen. The dependence of wide-doublet tyrosyl radical accumulation in PGHS isoform 1 on hydroperoxide stoichiometry, has been determined; this catalytically active radical is formed efficiently at stoichiometries </=1 after only 300 ms of reaction. This is consistent with intramolecular formation of the radical from PGHS Compound I but inconsistent with an alternative pathway involving reduction of Compound I to Compound II by a second hydroperoxide molecule. Results from stopped-flow studies indicate that the hydroperoxide level influences the rate of Compound II formation indirectly, via changes in the transient accumulation of Compound I, rather than by reducing Compound I. PGHS and soybean lipoxygenase reactions with 11,14-eicosadienoic acid (20:2) were also analyzed using a spectrophotometer cuvette fitted with an oxygen electrode to monitor lipid product formation and oxygen consumption simultaneously. The results show that the oxygen electrode signal is inherently dampened and thus underestimates the oxygen consumption rate; the discrepancy is much larger for the more rapidly accelerating PGHS reaction than for the lipoxygenase reaction. When correction is made for the electrode dampening, the ratio between the peak rates of oxygen consumption and lipid product formation was near unity for both PGHS and lipoxygenase, indicating a reaction stoichiometry of about 1 mol of O2 consumed/mol of 20:2 oxygenated for both enzymes. Separately, a stoichiometry of 0.9 mol of O2 consumed / mol oxygenated fatty acid was obtained when limiting amounts of 20:2 were reacted to completion with excess PGHS; the corresponding stoichiometry with arachidonic acid was 1.9. These O2/fatty acid stoichiometries are consistent with a dioxygenase mechanism for reaction of PGHS with both fatty acids and inconsistent with a mixed dioxygenase/monooxygenase mechanism proposed for the reaction with 20:2. The present conclusions reduce the complexity of the mechanisms that need to be considered for PGHS catalysis.