Prostaglandin H Synthase

Many cosubstrates for the peroxidase activity of prostaglandin H synthase-1 (PGHS-1) have been reported to produce a large (2–7-fold) increase in the cyclooxygenase velocity in addition to a substantial increase in the number of cyclooxygenase catalytic turnovers. The large stimulation of cyclooxygenase velocity has become an important criterion for evaluation of putative PGHS reaction mechanisms. This criterion has been a major weakness of branched-chain tyrosyl radical mechanisms, which correctly predict many other cyclooxygenase characteristics. Our computer simulations based on a branched-chain mechanism indicated that the uncorrected oxygen electrode signals commonly used to monitor activity can seriously overestimate the effects of cosubstrate on cyclooxygenase velocity. The simulation results prompted re-examination of the effect of several cosubstrates (phenol, acetaminophen,N,N,N′,N′-tetramethylphenylenediamine, and Trolox) on PGHS-1 cyclooxygenase velocity. Cyclooxygenase kinetics were examined at reduced temperature or elevated pH, where the oxygen electrode signal can be corrected to provide reliable oxygen consumption trajectories. The cosubstrates produced only a slight (10–60%) stimulation of the cyclooxygenase velocity. Peroxidase cosubstrates thus have a much smaller stimulatory effect on cyclooxygenase velocity than previously reported. This corrects a longstanding misperception of cosubstrate effects, provides more realistic kinetic constraints on PGHS mechanisms, and removes what was a major deficiency of branched-chain tyrosyl radical mechanisms.