Mechanistic studies on the inhibition of stromelysin by a peptide phosphonamidate

We have investigated the inhibition of the human matrix metalloproteinase stromelysin (SLN) by the peptide phosphonamidate, phthaloyl-N-(CH2)4-P(O2-)-Ile-(beta-naphthyl)Ala-NH-CH3, and find that it is a potent, slow-binding inhibitor of SLN with kon = 2.7 x 10(4) M-1 sec-1, koff = 1.9 x 10(-4) sec-1, and Ki = 7 nM (pH 5.0, 25 degrees C). To probe the mechanism of inhibition, we determined pH-dependencies and solvent deuterium isotope effects. pH-dependencies of the kinetic parameters for inhibition are complex but reflect greater inhibitory potency at lower pH and suggest a mechanism for inhibition that involves the same active site groups as are involved in catalysis. The solvent isotope on kon (kon, H2O/Kon,D2O) is normal and equals 1.5 +/- 0.1. Together with the pH-dependence of inhibition, this value suggests that kon is rate-limited by a process that involves general-acid/general-base catalysis. We propose that kon is rate-limited by general-acid catalyzed ligand exchange of inhibitor for the zinc-bound water molecule.