MECHANISTIC STUDIES ON THE HUMAN MATRIX METALLOPROTEINASE STROMELYSIN

To probe the mechanism of stromelysin (SLN)-catalyzed peptide hydrolysis, we determined the pH dependence of k(c)/K(m) and solvent deuterium isotope effects on k(c) and k(c)/K(m). pH dependencies of k(c)/K(m) were determined for the SLN-catalyzed hydrolysis of three peptides: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Nle-NH2, Arg-Pro-Ala-Pro-Gln-Gln-Phe-Phe-Gly-Leu-NleNH2, and N-acetyl-Arg-Pro-Ala-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Nle-NH2 (cleavage at Gln-Phe bond). The pH dependencies are all bell-shaped with shoulders that extend from pH 7.5 to 8.5. The existence of a shoulder indicates that the reaction mechanism involves at least two routes to products. These curves are governed by three proton ionizations with pK(a) values of 5.4, 6.1, and 9.5. The solvent isotope effect measurements provided the following values: D(k(c)/K(m)) = 0.80 +/- 0.05 and D(k(c)) = 1.58 +/- 0.05. That D(k(c)/K(m)) and D(k(c)) are different suggests that the rate-limiting transition states for the processes governed by k(c)/K(m) and k(c) cannot be the same. We use these results, together with analogy to thermolysin catalysis, to develop a mechanism for SLN catalysis.