Chemical modification of prostaglandin endoperoxide synthase by N‐acetylimidazol

Abstract

Prostaglandin H synthase apoprotein, without its prosthetic heme group, was inactivated by Nacetylimidazole under conditions typical for the O-acetylation of tyrosyl residues. A spontaneous reactivation occurred above pH 7.5 at 22 "C, which indicated spontaneous hydrolysis of acetylated residues. Below pH 7.5, where stable inactivation was observed, reactivation was achieved by reaction with hydroxylamine. Both enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, were inactivated and reactivated simultaneously and to the same extent. In contrast to the apoprotein, the holoenzyme with heme was not inactivated by N-acetylimidazole. The number of acetyl groups, as determined as hydroxamate after the reaction with hydroxylamine at pH 8.2, was 2.5 k 0.4 for the apoprotein and 1 .O If: 0.24 for the holoenzyme. The specific binding of heme as the prosthetic group was no longer observed by EPR (signals at g = 6.7 and 5.3) when hemin was added to the N-acetylimidazole-reacted apoprotein. Treatment of N-acetylimidazole-reacted apoprotein with hydroxykamine restored the specific binding of heme. The N-acetylimidazole-reacted apoprotein supplemented with hemin and reacted with hydroperoxides, neither showed electronic absorption spectra of higher oxidation states nor an EPR doublet signal due to a tyrosyl radical.These results demonstrate that heme protects against the inactivating modification by Nacetylimidazole and that this modification prevents binding of the prosthetic heme group necessary for both enzymic activities. The absence of the prosthetic heme group explains the concomitant loss of cyclooxygenase and peroxidase activities, as well as the absence of higher oxidation states and the tyrosyl radical. We suggest that the acetylation of a residue in the heme pocket, most probably a tyrosine, although a histidine cannot be definitely disproved, exerts the inhibiting effect. This residue could be the axial ligand of the heme or in close contact to the heme. The results also show that the inhibition by N-acetylimidazole does not involve the acetylation of Ser530 which causes the inhibition by acetylsalicylic acid of cyclooxygenase. [The numbering of amino acids in ovine prostaglandin H synthase is according to DeWitt, D.

Inactivation of prostaglandin endoperoxide synthase by acylating derivatives of indomethacin

“…Reversal of inactivation by hydroxylamine rules out serine and lysine residues (Riordan et al, 1965). Reversal of inactivation by incubation at elevated pH's is similar to the behavior of PGH synthase inactivated by NAI, which Scherer et al attributed to acetyltyrosine (Scherer et al, 1992). However, the instability of acylated PGH synthase to chromatography seems to exclude tyrosine as a potential site for acylation.…”

“…Previous reports from our laboratory and Ruf s laboratory demonstrated that NAI inactivates the cyclooxygenase and peroxidase activities of ovine PGH synthase (Wells & Marnett, 1992;Scherer et al, 1992). The presence of the heme prosthetic group protected the protein from inactivation, and acetylated protein was not reconstituted by heme (Scherer et al, 1992). Thus, it appeared that NAI was reacting with an amino acid residue near the heme binding site and preventing reconstitution of the apoprotein.…”

In Vitro Modification of Bacterial Cyanophycin and Cyanophycin Dipeptides Using Chemical Agents Towards Novel Variants of the Biopolymer

“…It also acetylates thiol groups, but with lower reactivity (Herriott 1947). N-acetylimidazole can be used for acetylation of proteins, but has a lower specificity and reactivity for amino groups, and it is mainly used for acetylation of tyrosine residues (Pal et al 1999;Riordan et al 1965;Scherer et al 1992;Zhang et al 2005). Acetylation of compounds can be interesting for various reasons.…”

Protein‐radical enzymes

“…Much evidence now indicates that the tyrosyls are not involved directly in the cyclooxygenase reaction, but instead play a role in the suicide process that accompanies catalysis [42][43][44], although this point is still controversial [46].…”