Mechanism for irreversible self-deactivation of prostaglandin synthetase.
It has been shown that prostaglandin (PG) cyclooxygenase is irreversibly self-deactivated during the oxygenation of arachidonic acid (Smith, W. L., and Lands, W.E.M. (1972) Biochemistry II, 3273-3285). Using several experimental approaches and an enzyme preparation which was highly active without artificial stimulation, we have extensively investigated the mechanism of this deactivation process. During the generation of PGH2 from arachidonic acid, oxidizing equivalents were released and the reductive breakdown of PGG2 was found to deactivate the cyclooxygenase. The cyclooxygenase-catalyzed metabolism of both arachidonic acid and PGG2 generated radicals which were scavenged by phenol. Both phenol and methional (scavengers of oxygen-centered radicals) promoted the formation of PGH2 at the expense of PGG2 and increased the initial rate and the extent of reaction prior to deactivation of the cyclooxygenase. Hence, it appears that the cyclooxygenase was irreversibly self-deactivated during the formation of PGH2 from arachidonic acid, due to its oxidation by oxygen-centered radicals formed as a result of the reductive breakdown of the hydroperoxide on PGG2. Some experiments with dithiothereitol and N-ethylmaleimide suggested that the enzyme may contain a disulfide at the active site. A mechanism has been devised which accounts for the self-deactivation phenomenon, the effect of phenol and methional, the disulfide at the active site, and the pathway of substrate oxygenation.
D000332:Aerobiosis; D000447:Aldehydes; D000693:Anaerobiosis; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D001665:Binding Sites; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010636:Phenols / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011485:Protein Binding; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep; D013438:Sulfhydryl Compounds
D000447:Aldehydes; D001095:Arachidonic Acids; D010636:Phenols; D013438:Sulfhydryl Compounds; C008390:methional; D006899:Mixed Function Oxygenases; D011451:Prostaglandin-Endoperoxide Synthases; D005033:Ethylmaleimide; D004229:Dithiothreitol