A fluorescence resonance energy transfer method for measuring the binding of inhibitors to stromelysin

A sensitive fluorescence resonance energy transfer method was developed for the direct measurement of the dissociation constants of stromelysin inhibi tors. The method is applied to the thiadiazole class of stromelysin inhibit ors and it takes advantage of the fact that, upon binding to the active sit e of enzyme, the thiadiazole ring, with its absorbance centered at 320 nm, is able to quench the fluorescence of the tryptophan residues surrounding t he catalytic site. The changes in fluorescence are proportional to the occu pancy of the active site: Analysis of the fluorescence versus inhibitor con centration data yields dissociation constants that are in agreement with th e corresponding competitive inhibitory constants measured by a catalytic ra te assay. The affinity of nonthiadiazole inhibitors of stromelysin-such as hydroxamic acids and others-can be determined from the concentration-depend ent displacement of a thiadiazole of known affinity. Using this displacemen t method, we determined the affinities of a number of structurally diverse inhibitors toward stromelysin, Since the three tryptophan residues located in the vicinity of the active site of stromelysin are conserved in gelatina se and collagenase, the method should also be applicable to inhibitors of o ther matrix metalloproteinases. (C) 1999 Academic Press.