Chemical modification of prostaglandin endoperoxide synthase by N-acetylimidazole. Effect on enzymic activities and EPR spectroscopic properties.
Prostaglandin H synthase apoprotein, without its prosthetic heme group, was inactivated by N-acetylimidazole under conditions typical for the O-acetylation of tyrosyl residues. A spontaneous reactivation occurred above pH 7.5 at 22 degrees 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 +/- 0.4 for the apoprotein and 1.0 +/- 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 hydroxylamine 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 N-acetylimidazole 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 effects. 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. L. and Smith, W. L. (1988) Proc. Natl Acad. Sci. USA 85, 1412-1416 including a signal peptide of 24 residues which is missing in the processed protein.
Scherer
H J
HJ
Fraunhofer Institut für Biomedizinische Technik, Universität des Saarlandes, St. Ingbert, Federal Republic of Germany.
Karthein
R
R
Strieder
S
S
Ruf
H H
HH
D000107:Acetylation; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D011451:Prostaglandin-Endoperoxide Synthases / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep
D001051:Apoenzymes; D006898:Hydroxylamines; D007093:Imidazoles; D019811:Hydroxylamine; D011451:Prostaglandin-Endoperoxide Synthases; C012160:N-acetylimidazole