Comparison of branched-chain and tightly coupled reaction mechanisms for prostaglandin H synthase

Abstract

Two types of mechanisms have been proposed to account for the combination of peroxidase and cyclooxygenase activities in prostaglandin H synthase (PGHS). One, a branched-chain mechanism [Dietz, R., et al. (1988) Eur. J. Biochem. 171, 321-328], postulates that the cyclooxygenase reaction propagates essentially independently of peroxidase catalysis. The second, a tightly coupled mechanism [Bakovic, M., & Dunford, H. B. (1994) Biochemistry 33, 6475-6482], postulates that peroxidase catalysis is an integral part of cyclooxygenase propagation. Qualitative and quantitative predictions from the two mechanisms have been compared with several observed characteristics of the PGHS reaction with arachidonate, including the ability to accumulate PGG2 and oxidized enzyme intermediates, the stoichiometry between cosubstrate and fatty acid consumption, and the hydroperoxide activator requirement. The observed characteristics, particularly the accumulation of micromolar levels of PGG2 even in the presence of cosubstrate and the stoichiometry between cosubstrate oxidation and fatty acid oxygenation of less than 1.3 (compared to a theoretical maximum of 2.0), were largely consistent with predictions from the branched-chain mechanism, but contradicted important predictions of the tightly coupled mechanism. These results indicate that PGHS catalysis is more accurately described by the branched-chain mechanism than by the tightly coupled mechanism.

Role of Asn-382 and Thr-383 in Activation and Inactivation of Human Prostaglandin H Synthase Cyclooxygenase Catalysis

“…The predicted cyclooxygenase reaction time course was monitored from the changes in arachidonate concentration. If desired, the corresponding oxygen electrode reaction time course was calculated to reflect the dampening effect of the electrode membrane (23). Glutathione peroxidase activity was calculated from a MichaelisMenten expression involving the ratio of added cGPx units to added cyclooxygenase units, the number of added cyclooxygenase units, the peroxide concentration, and the K m of cGPx for peroxide.…”

Prostaglandin H Synthase

“…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.by guest on May 7, 2018 http://www.jbc.org/ Downloaded from 3 The cyclooxygenase activity of prostaglandin H synthase isoforms 1 and 2 1 is a key control point in the biosynthesis of all prostanoid lipid mediators (1,2). Besides the cyclooxygenase activity, both PGHS-1 and -2 have a heme-dependent peroxidase activity (1).…”

“…by guest on May 7, 2018 http://www.jbc.org/ Downloaded from 3 The cyclooxygenase activity of prostaglandin H synthase isoforms 1 and 2 1 is a key control point in the biosynthesis of all prostanoid lipid mediators (1,2). Besides the cyclooxygenase activity, both PGHS-1 and -2 have a heme-dependent peroxidase activity (1).…”

“…The protective action of cellular reductants greatly increases the number of catalytic turnovers before cyclooxygenase selfinactivation, thereby increasing the capacity for synthesis of potent lipid mediators (14). For its part, the large stimulation of cyclooxygenase velocity by cosubstrate has become a defining characteristic of catalytic behavior and an important criterion for evaluation of potential PGHS reaction mechanisms (11,13,15 (15,27,28). However, one perceived deficiency of branched-chain mechanisms has been an inability to predict significant stimulation of cyclooxygenase velocity by cosubstrates without ad hoc assumptions (15,17).…”

“…For its part, the large stimulation of cyclooxygenase velocity by cosubstrate has become a defining characteristic of catalytic behavior and an important criterion for evaluation of potential PGHS reaction mechanisms (11,13,15 (15,27,28). However, one perceived deficiency of branched-chain mechanisms has been an inability to predict significant stimulation of cyclooxygenase velocity by cosubstrates without ad hoc assumptions (15,17). Recent studies have extended the basic branched-chain mechanism for PGHS to include steps for peroxidase self-inactivation (29).…”