title: Mechanistic studies on the human matrix metalloproteinase stromelysin.
abstract: To probe the mechanism of stromelysin (SLN)-catalyzed peptide hydrolysis, we determined the pH dependence of kc/Km and solvent deuterium isotope effects on kc and kc/Km. pH dependencies of kc/Km 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 pKa values of 5.4, 6.1, and 9.5. The solvent isotope effect measurements provided the following values: D(kc/Km) = 0.80 +/- 0.05 and D(kc) = 1.58 +/- 0.05. That D(kc/Km) and D(kc) are different suggests that the rate-limiting transition states for the processes governed by kc/Km and kc cannot be the same. We use these results, together with analogy to thermolysin catalysis, to develop a mechanism for SLN catalysis.
authors.affiliation: Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065.
authors.forename: R K
authors.initials: RK
authors.lastname: Harrison
authors.forename: B
authors.initials: B
authors.lastname: Chang
authors.forename: L
authors.initials: L
authors.lastname: Niedzwiecki
authors.forename: R L
authors.initials: RL
authors.lastname: Stein
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D003903:Deuterium; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D012997:Solvents
chemical_list: D009842:Oligopeptides; D012997:Solvents; D003903:Deuterium; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3