title: Metal and pH dependence of heptapeptide catalysis by human matrilysin.
abstract: Human matrilysin devoid of its propeptide is expressed in Escherichia coli and purified to homogeneity by heparin chromatography after refolding of the guanidine hydrochloride solubilized protein. Matrilysin autolytically removes its N-terminal tripeptide Met-Tyr-Ser during the refolding process. The enzyme contains 1.91 +/- 0.08 zinc atoms/mol of protein and retains full activity when stored several months at 4 degrees C. It hydrolyzes the fluorescent substrate Dns-PLALWAR at the Ala-Leu bond with a kcat of 3.1 s-1 and K(m) of 1.8 x 10(-5) M at pH 7.5, 37 degrees C, values closely similar to those for the matrilysin produced by activation of the Chinese hamster ovary and E. coli-expressed promatrilysin. The properties of this form of matrilysin demonstrate that the propeptide is not essential for proper folding or stability of the enzyme but likely determines the N-terminal amino acid of the mature enzyme. The pH dependence of kcat/K(m) for Dns-PLALWAR shows that matrilysin has a broad pH optimum (5.0-9.0) and the pKa values obtained are 4.3 and 9.6 at 25 degrees C. The activity is inhibited by several metal binding agents including 1, 10-phenanthroline, OP, but not by the nonchelating isomer, 1,7-phenanthroline. OP inhibits instantaneously by likely forming a transient ternary enzyme.metal.chelator complex. The zinc atom is then removed from the protein in a time-dependent manner. In agreement with the kinetic studies, dialysis in the presence of OP and CaCl2 removes only the catalytic zinc atom. The monozinc enzyme can be reactivated to 90%, 56%, 27%, and 17% of the native activity by addition of zinc, manganese, nickel, and cobalt, respectively. Cadmium, on the other hand, forms an inactive Cd/Zn hybrid. The differences in the chelator accessibility properties of the two zinc sites can thus be exploited to yield metallohybrids of matrilysin.
authors.affiliation: Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: J
authors.initials: J
authors.lastname: Cha
authors.forename: M V
authors.initials: MV
authors.lastname: Pedersen
authors.forename: D S
authors.initials: DS
authors.lastname: Auld
mesh_terms: D000595:Amino Acid Sequence; D002104:Cadmium / Q000032:analysis; D002614:Chelating Agents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D020783:Matrix Metalloproteinase 7; D008666:Metalloendopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019216:Metals, Heavy / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000032:analysis
chemical_list: D002614:Chelating Agents; D019216:Metals, Heavy; D010446:Peptide Fragments; D010618:Phenanthrolines; D011994:Recombinant Proteins; D002104:Cadmium; D008666:Metalloendopeptidases; D020783:Matrix Metalloproteinase 7; D015032:Zinc; C025205:1,10-phenanthroline