THE CONSTITUENT TRYPTOPHANS AND BISANS AS FLUORESCENT-PROBES OF THE ACTIVE-SITE AND OF A SECONDARY BINDING-SITE OF STROMELYSIN-1 (MMP3)

The active site of the catalytic domain of stromelysin-1 (matrix metal loproteinase-3, MMP-3) was probed by fluorescence quenching, lifetime, and polarization of its three intrinsic tryptophans and by the enviro nmentally sensitive fluorescent reporter molecule bisANS. Wavelength-d ependent acrylamide quenching identified three distinct emitting trypt ophan species, only one of which changes its emission and fluorescence lifetime upon binding of the competitive inhibitor Batimastat. Signif icant changes in the tryptophan fluorescence polarization occur upon b inding by any of the three hydroxamate inhibitors Batimastat, CAS10838 3-58-0, and Celltech CT1418, all of which bind in the P2'-P3' region o f the active site. In contrast, the inhibitor CGS27023A, which is thou ght to bind in the P1-P1' region, does not induce any change in trypto phan fluorescence polarization. The use of the fluorescent probe bisAN S revealed the existence of an auxiliary binding site extrinsic to the catalytic cleft. BisANS acts as a competitive inhibitor of stromelysi n with a dissociation constant of K-i = 22 mu M. In addition to this b inding to the active site, it also binds to the auxiliary site with a dissociation constant of 3.40 +/- 0.17 mu M. The auxiliary site is ope n, hydrophobic, and near the fluorescing tryptophans. The binding of b isANS to the auxiliary site is greatly enhanced by Batimastat, but not by the other competitive inhibitors tested.