Kinetics of the interaction of nonsteroidal antiinflammatory drugs with prostaglandin endoperoxide synthase-1 studied by limited proteolysis.

Kalgutkar AS, Crews BC, Marnett LJ
Biochemistry. 1996 35 (28): 9076-82

PMID: 8703911 · DOI:10.1021/bi9605752

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.

MeSH Terms (15)

Anti-Inflammatory Agents, Non-Steroidal Arachidonic Acid Cyclooxygenase Inhibitors Hemin Ibuprofen Kinetics Molecular Structure Nitrobenzenes Prostaglandin-Endoperoxide Synthases Prostanoic Acids Protein Binding Protein Conformation Sulfonamides Thiazoles Trypsin

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