Stoichiometry and kinetics of the interaction of prostaglandin H synthase with anti-inflammatory agents.

Abstract

We have examined the kinetics, stoichiometry, and chemical nature of the interaction of three anti-inflammatory agents (indomethacin, flurbiprofen, and meclofenamic acid) with pure ovine prostaglandin H synthase. The kinetics of the interaction with the synthase for each of the three agents, monitored by the decrease in cyclooxygenase activity, was consistent with the model proposed by Rome and Lands (Rome, L.H., and Lands, W.E.M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4863-4865): a rapid and reversible initial binding, followed by a first-order decay of the synthase-inhibitor complex. A relatively stable form of the cyclooxygenase, which had 4-10% of the initial activity, was the eventual product of this decay process. The dissociation constants evaluated for the initial binding were 1.7 +/- 1.5 microM for indomethacin, 0.2 +/- 0.1 microM for flurbiprofen, and 0.08 +/- 0.06 microM for meclofenamic acid. The values of the first order rate constants for the subsequent decay process were 14.9 +/- 11.3 min-1 for indomethacin, 3.4 +/- 0.7 min-1 for meclofenamic acid, and 16.6 +/- 6.2 min-1 for flurbiprofen. In repeated titrations of the cyclooxygenase with the three agents, 1.3 +/- 0.3 mol of indomethacin, 1.2 +/- 0.1 mol of meclofenamic acid, and 1.2 +/- 0.1 mol of S-(+)-flurbiprofen/mol of synthase dimer were found to result in maximal inhibition of the enzyme. Racemic flurbiprofen required 2.4 +/- 0.3 mol/mol synthase dimer for full effect, and the R-(-)-isomer was not inhibitory. Inhibition of the cyclooxygenase activity by these agents thus appears to result from a stereospecific binding to only one of the subunits of the synthase. Intact indomethacin could be recovered quantitatively after prolonged incubation (in stoichiometric quantities) with the synthase had resulted in maximal inhibition of the cyclooxygenase activity. The time-dependent effect of indomethacin on the cyclooxygenase is thus likely to involve a conformational change in the synthase rather than a covalent interaction.