Differential modification of cyclo-oxygenase and peroxidase activities of prostaglandin endoperoxidase synthase by proteolytic digestion and hydroperoxides.
Prostaglandin endoperoxide synthase (PES, EC 1.14.99.1) catalyse the conversion of arachidonic acid into prostaglandin H2. The enzyme is a 140 kDa homodimer which contains both a cyclo-oxygenase activity (converting arachidonate into prostaglandin G2) and peroxidase activity (reducing prostaglandin G2 to H2). PES undergoes rapid self-inactivation during oxygenation of arachidonate to prostaglandin H2 in vitro. The previously reported cDNA-derived amino acid sequence indicates numerous sites for trypsin or thrombin cleavage. Most of these sites must be inaccessible, since these enzymes cleave only at Arg253. The enzyme appears to be a self-adherent and highly folded molecule, since after cleavage it retains its functional assembly and its homodimer size of 140 kDa, as well as its overall enzymic activity. Only under denaturing conditions (e.g. SDS/PAGE) can the proteolytic peptides be demonstrated: a 38 kDa C-terminal fragment containing the aspirin-derived-acetyl-binding ability, and a 33 kDa N-terminal fragment. In the present studies we investigated whether the two enzymic activities of PES can be differentially manipulated by proteolytic cleavage or by substrate (arachidonate) self-inactivation. The results indicated that, during arachidonate oxygenation by PES, the cyclooxygenase activity is selectively inactivated, whereas the peroxidase activity is essentially retained. By contrast, thrombin or trypsin cleavage of pure PES or microsomal PES (to yield the 38 and 33 kDa peptide fragments) inactivated the peroxidase, but not the cyclo-oxygenase. Taken together, these results suggest the presence of separate cyclo-oxygenase and peroxidase structural domains on the enzyme.
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Department of Pharmacology, Washington University School of Medicine, St. Louis 63110.
Needleman
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D000818:Animals; D004789:Enzyme Activation; D008861:Microsomes / Q000201:enzymology; D010447:Peptide Hydrolases / Q000378:metabolism; D010544:Peroxidases / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D012756:Sheep; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010544:Peroxidases; D011451:Prostaglandin-Endoperoxide Synthases; D010447:Peptide Hydrolases; D014357:Trypsin; D013917:Thrombin