Comparison of the peroxidase reaction kinetics of prostaglandin H synthase-1 and-2

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) each have a perox idase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide, The hydroperoxide initiator requirement for PGHS-2 cycloo xygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this di fference may contribute to the distinct control of cellular prostanoid synt hesis by the two isoforms. We compared the kinetics of the initial peroxida se steps in PGHS-1 and -2 to quantify mechanistic differences between the i soforms that might contribute to the difference in cyclooxygenase initiatio n efficiency, The kinetics of formation of Intermediate I (an Fe(IV) specie s with a porphyrin free radical) and Intermediate II (an Fe(IV) species wit h a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygena se catalysis) were monitored at 4 degrees c by stopped flow spectrophotomet ry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetra enoic acid, the rate constant for Intermediate I formation (k(1)) was 2.3 x 10(7) M-1 s(-1) for PGHS-1 and 2.5 x 10(7) M-1 s(-1) for PGHS-2, indicatin g that the isoforms have similar initial reactivity with this lipid hydrope roxide, For PGHS-1, the rate of conversion of Intermediate I to Intermediat e II (k(2)) became the limiting factor when the hydroperoxide level was inc reased, indicating a rate constant of 10(2)-10(3) s(-1) for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition b etween Intermediates I and II was not rate-limiting even at the highest hyd roperoxide concentrations tested, indicating that the k(2) value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that f aster formation of the active cyclooxygenase species (Intermediate II) or i ncreased stability of the active species increases the resistance of the cy clooxygenase to inhibition by the intracellular hydroperoxide scavenger; gl utathione peroxidase, Kinetic differences between the PGHS isoforms in form ing or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.