Reaction Of Prostaglandin Research Articles

Cyclooxygenase Competitive Inhibitors Alter Tyrosyl Radical Dynamics in Prostaglandin H Synthase-2

Reaction of prostaglandin H synthase (PGHS) isoforms 1 or 2 with peroxide forms a radical at Tyr385 that is required for cyclooxygenase catalysis and another radical at Tyr504, whose function is unknown. Both tyrosyl radicals are transient and rapidly dissipated by reductants, suggesting that cyclooxygenase catalysis might be vulnerable to suppression by intracellular antioxidants. Our initial hypothesis was that the two radicals are in equilibrium and that their proportions and stability are altered upon binding of fatty acid substrate. As a test, we examined the effects of three competitive inhibitors (nimesulide, flurbiprofen, and diclofenac) on the proportions and stability of the two radicals in PGHS-2 pretreated with peroxide. Adding nimesulide after ethyl peroxide led to some narrowing of the tyrosyl radical signal detected by EPR spectroscopy, consistent with a small increase in the proportion of the Tyr504 radical. Neither flurbiprofen nor diclofenac changed the EPR line width when added after peroxide. In contrast, the effects of cyclooxygenase inhibitors on the stability of the preformed tyrosyl radicals were dramatic. The half-life of total tyrosyl radical was 4.1 min in the control, >10 h with added nimesulide, 48 min with flurbiprofen, and 0.8 min with diclofenac. Stabilization of the tyrosyl radicals was evident even at substoichiometric levels of nimesulide. Thus, the inhibitors had potent, structure-dependent, effects on the stability of both tyrosyl radicals. This dramatic modulation of tyrosyl radical stability by cyclooxygenase site ligands suggests a mechanism for regulating the reactivity of PGHS tyrosyl radicals with cellular antioxidants.

Identification of Two Cyclooxygenase Active Site Residues, Leucine 384 and Glycine 526, That Control Carbon Ring Cyclization in Prostaglandin Biosynthesis

The cyclooxygenase (COX) reaction of prostaglandin (PG) biosynthesis begins with the highly specific oxygenation of arachidonic acid in the 11R configuration and ends with a 15S oxygenation to form PGG2. To obtain new insights into the mechanisms of stereocontrol of oxygenation, we mutated active site residues of human COX-2 that have potential contacts with C-11 of the reacting substrate. Although the 11R oxygenation was not perturbed, changing Leu-384 (into Phe, Trp), Trp-387 (Phe, Tyr), Phe-518 (Ile, Trp, Tyr), and Gly-526 (Ala, Ser, Thr, Val) impaired or abrogated PGG2 synthesis, and typically 11R-HETE was the main product formed. The Gly-526 and Leu-384 mutants formed, in addition, three novel products identified by LC-MS, NMR, and circular dichroism as 8,9-11,12-diepoxy-13R-(or 15R)-hydro(pero)xy derivatives of arachidonic acid. Mechanistically, we propose these arise from a free radical intermediate in which a C-8 carbon radical displaces the 9,11-endoperoxide O-O bond to yield an 8,9-11,12-diepoxide that is finally oxygenated stereospecifically in the 13R or 15R configuration. Formation of these novel products signals an arrest in the normal course of prostaglandin synthesis just prior to closing of the 5-membered carbon ring, and points to a crucial role for Leu-384 and Gly-526 in the correct positioning of the reacting fatty acid intermediate. Some of the Gly-526 and Leu-384 mutants catalyzed both formation of PGG2 (with the normal 15S configuration) and the 13R- or 15R-oxygenated diepoxides. This result suggests that oxygenation specificity can be determined by the orientation of the reacting fatty acid radical and is not a predetermined outcome based solely on the structure of the cyclooxygenase active site.

Similarities in the optical spectra of prostaglandin H synthase during its cyclooxygenase and peroxidase reactions

The spectral behavior of the enzyme prostaglandin H synthase was studied in the Soret region under conditions that permitted comparison of enzyme intermediates involved in peroxidase and cyclooxygenase activities. First, the peroxidase activity was examined. The enzyme's spectral behavior upon reacting with 5-phenyl-pent-4-enyl-1-hydroperoxide was different depending on the presence or absence of the reducing substrate, phenol. In the reaction of prostaglandin H synthase with the peroxide in the absence of phenol, formation of the enzyme intermediate compound I is observed followed by partial conversion to compound II and then by enzyme bleaching. In the reaction with both peroxide and phenol the absorbance decreases and a steady-state spectrum is observed which is a mixture of native enzyme and compound II. The steady state is followed by an increase in absorbance back to that of the native enzyme with no bleaching. The difference can be explained by the reactivity of phenol as a reducing substrate with the prostaglandin H synthase intermediate compounds. Cyclooxygenase activity with arachidonic acid could not be examined in the absence of diethyldithiocarbamate because extensive bleaching occurred. In the presence of diethyldithiocarbamate, enzyme spectral behavior similar to that seen in the reaction of the peroxide and phenol was observed. The similarity of the spectra strongly suggests that the enzyme intermediates involved in both the peroxidase and cyclooxygenase reactions are the same.

Higher oxidation states of prostaglandin H synthase. Rapid electronic spectroscopy detected two spectral intermediates during the peroxidase reaction with prostaglandin G2.

The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1 degree C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1 = 1.4 x 10(7) M-1 s-1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+.FeIVO]. Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2 = 65 s-1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome-c peroxidase, was assigned to a two-electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+. which was formed by an intramolecular electron transfer from tyrosine to the porphyrin-pi-cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction.

Prostaglandin G2 levels during reaction of prostaglandin H synthase with arachidonic acid

Prostaglandin H synthase catalyzes the formation of prostaglandin (PG) G2 from arachidonic acid (cyclooxygenase activity), and also the reduction of PGG2 to PGH2 (peroxidase activity). The ability of the pure synthase to accumulate the hydroperoxide, PGG2, under conditions allowing the concurrent function of both catalytic activities was investigated. The peroxidase velocity was continuously determined from the absorbance increases at 611 nm that accompanied oxidation of a peroxidase cosubstrate, N,N,N′N′-tetramethyl-phenylenediamine, and PGG2 concentrations were calculated from the peroxidase velocities and the peroxidase Vmax and Km values. Cyclooxygenase velocities were than calculated from the changes in PGG2. Parallel reactions monitored by the use of radiolabelled arachidonate or with a polarographic oxygen electrode were used to confirm the calculated PGG2 levels and the cyclooxygenase velocities. The concentration of PGG2 was found to follow a transient course as the reaction of the synthase progressed, rapidly rising to a maximum of 0.7μM in the first 10 s, and then declining slowly, reaching 0.1 μM after 60 s. The maximal level of PGG2 achieved during the reaction was constant at about 0.7 μM with higher amounts of added cyclooxygenase capacity (0.3–0.6 μM PGG2/s) but was only about 0.4 μM when the added cyclooxygenase capacity was 0.1 μM PGG2/s. The peroxidase was found to lose only 30% of its activity after 90 s, a point where the cyclooxygenase was almost completely inactive. These results support the concept of a burst of catalytic action from the cyclooxygenase and a reactive, more sustained, catalytic action from the peroxidase during the reaction of the synthase with arachidonic acid.