Topography of prostaglandin H synthase. Antiinflammatory agents and the protease-sensitive arginine 253 region.
Prostaglandin H synthase catalyzes two reactions: the bis-dioxygenation of arachidonic acid to form prostaglandin G2 (cyclooxygenase activity), and the reduction of hydroperoxides to the corresponding alcohols (peroxidase activity). The cyclooxygenase activity can be selectively inhibited by many nonsteroidal antiinflammatory agents including indomethacin. In the native synthase, there is a single prominent protease-sensitive region, located near Arg253; binding of the heme prosthetic group makes the synthase resistant to proteases. To investigate the spatial relationship between the area of the synthase which interacts with indomethacin and the protease-sensitive region, the effects of indomethacin and similar agents on the protease sensitivity of the two enzymatic activities and of the synthase polypeptide were examined. Incubation of the synthase apoenzyme with trypsin (3.6% w/w) resulted in the time-dependent coordinate loss (75% at 1 h) of both enzymatic activities and the cleavage (85% at 1 h) of the 70-kDa subunit into 38- and 33-kDa fragments, indicating that proteolytic cleavage of the polypeptide at Arg253, destroyed both activities of the synthase simultaneously. Indomethacin, (S)-flurbiprofen, or meclofenamate (each at 20 microM) rendered both activities and the synthase polypeptide (at 5 microM subunit) resistant to attack by trypsin or proteinase K; these agents also inhibited the cyclooxygenase activity of the intact synthase. Two reversible cyclooxygenase inhibitors, ibuprofen and flufenamate, also made both of the activities and the synthase polypeptide more resistant to trypsin. Titration of the apoenzyme with indomethacin (0-3 mol/mol of synthase dimer) resulted in proportional increases in the inhibition of the cyclooxygenase and in the resistance to attack by trypsin. (R)-Flurbiprofen did not increase the resistance to protease or appreciably inhibit the cyclooxygenase. These results suggest that the same stereospecific interaction of these agents with the synthase that produced inhibition of the cyclooxygenase led to a decreased accessibility of the Arg253 region to proteases. Aspirin treatment made the synthase less resistant to trypsin; aspirin-treated synthase became more resistant to trypsin when it was incubated with indomethacin before addition of the protease. The presence of 50 microM arachidonate during digestion of apoenzyme or aspirin-treated apoenzyme with trypsin did not decrease the cleavage of the synthase subunit.(ABSTRACT TRUNCATED AT 400 WORDS)
Kulmacz
R J
RJ
Department of Biological Chemistry, University of Illinois, Chicago 60612.
D000107:Acetylation; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D002942:Circular Dichroism; D016861:Cyclooxygenase Inhibitors; D007213:Indomethacin / Q000494:pharmacology; D010447:Peptide Hydrolases; D010544:Peroxidases / Q000037:antagonists & inhibitors; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D012756:Sheep; D013329:Structure-Activity Relationship; D014357:Trypsin
D000894:Anti-Inflammatory Agents, Non-Steroidal; D001095:Arachidonic Acids; D016861:Cyclooxygenase Inhibitors; D016718:Arachidonic Acid; D001120:Arginine; D010544:Peroxidases; D011451:Prostaglandin-Endoperoxide Synthases; D010447:Peptide Hydrolases; D014357:Trypsin; D007213:Indomethacin