Investigation of the binding of isoform-selective inhibitors to prostaglandin endoperoxide synthases using fluorescence spectroscopy.

Abstract

Prostaglandin endoperoxide synthase (PGHS) is a heme protein that catalyzes the committed step in prostaglandin and thromboxane biosynthesis. Two isoforms of PGHS exist, a constitutive form termed PGHS-1 and an inducible form termed PGHS-2. We report here fluorescence resonance energy transfer analysis of isoform-selective inhibitors interacting with PGHS-1 and PGHS-2. By measuring fluorescence quenching due to the energy transfer of the inhibitor fluorescence to the heme prosthetic group of PGHS, we determined these inhibitors bind in the arachidonic acid substrate access channel with an R0 of 35 A for PGHS-1 with the PGHS-1 inhibitor and an R0 of 21 A for PGHS-2 with the PGHS-2 inhibitor. The observed fluorescence quenching is completely dynamic and dominated by quenching by the heme. Time-resolved results combined with molecular modeling determine the distance from the inhibitor to the heme moiety to be 20 A in PGHS-1 and 18 A in PGHS-2. Preliminary stopped-flow kinetic studies reveal that the rate of quenching is limited by a first-order protein transition, which is slow, and that bound inhibitor undergoes rapid exchange.