Kinetics of the interaction of nonsteroidal antiinflammatory drugs with prostaglandin endoperoxide synthase-1 studied by limited proteolysis.
Many nonsteroidal antiinflammatory agents (NSAIDs) bind to prostaglandin endoperoxide synthase (PGHS) and induce a conformational change in the PGHS apoprotein that renders it resistant to cleavage by trypsin at Arg277. In the present study, the trypsin protection assay was modified to permit detection of conformational changes at times as short as 5 s after the addition of inhibitor. The kinetics of the induction and reversal of trypsin resistance in apoPGHS-1 by a series of NSAIDs and isozyme-specific PGHS-1 and PGHS-2 inhibitors were determined. All compounds induced resistance to trypsin cleavage at a rapid rate. The conformational change induced by competitive inhibitors was reversed on prolonged incubation with trypsin (approximately 5 min). In contrast, the resistance induced by irreversible inhibitors was not lost during a 5 min incubation with trypsin. All of the selective PGHS-2 inhibitors protected against tryptic cleavage of apoPGHS-1 but did not inhibit the protein's cyclooxygenase activity. The results suggest that induction of trypsin resistance is a reflection of the initial association of reversible as well as irreversible inhibitors with the apoprotein.
Kalgutkar
A S
AS
A.B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Crews
B C
BC
Marnett
L J
LJ
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism / Q000494:pharmacology*; D016718:Arachidonic Acid / Q000494:pharmacology; D016861:Cyclooxygenase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006427:Hemin / Q000494:pharmacology; D007052:Ibuprofen / Q000494:pharmacology; D007700:Kinetics; D015394:Molecular Structure; D009578:Nitrobenzenes / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000378:metabolism*; D011466:Prostanoic Acids / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013449:Sulfonamides / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D000894:Anti-Inflammatory Agents, Non-Steroidal; D016861:Cyclooxygenase Inhibitors; D009578:Nitrobenzenes; D011466:Prostanoic Acids; C100833:SC 8076; C100834:SC 8092; D013449:Sulfonamides; D013844:Thiazoles; C075261:1-(2,2-dimethylhept-5-eno-7-yl)-2-((hexyloxy)methyl)-7-oxabicyclo(2.2.1)heptane; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D016718:Arachidonic Acid; D006427:Hemin; D011451:Prostaglandin-Endoperoxide Synthases; D014357:Trypsin; C012655:nimesulide; D007052:Ibuprofen