INVESTIGATION OF THE BINDING OF ISOFORM-SELECTIVE INHIBITORS TO PROSTAGLANDIN ENDOPEROXIDE SYNTHASES USING FLUORESCENCE SPECTROSCOPY

Prostaglandin endoperoxide synthase (PGHS) is a heme protein that cata lyzes 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 e nergy transfer analysis of isoform-selective inhibitors interacting wi th PGHS-1 and PGHS-2. By measuring fluorescence quenching due to the e nergy transfer of the inhibitor fluorescence to the heme prosthetic gr oup of PGHS, we determined these inhibitors bind in the arachidonic ac id substrate access channel with an R-0 of 35 Angstrom for PGHS-1 with the PGHS-1 inhibitor and an R-0 of 21 Angstrom for PGHS-2 with the PG HS-2 inhibitor. The observed fluorescence quenching is completely dyna mic and dominated by quenching by the heme. Time-resolved results comb ined with molecular modeling determine the distance from the inhibitor to the heme moiety to be 20 Angstrom in PGHS-1 and 18 Angstrom in PGH S-2. Preliminary stopped-flow kinetic studies reveal that the rate of quenching is limited by a first-order protein transition, which is slo w, and that bound inhibitor undergoes rapid exchange.