METAL AND PH-DEPENDENCE OF HEPTAPEPTIDE CATALYSIS BY HUMAN MATRILYSIN

Human matrilysin devoid of its propeptide is expressed in Escherichia coli and purified to homogeneity by heparin chromatography after refol ding of the guanidine hydrochloride solubilized protein. Matrilysin au tolytically removes its N-terminal tripeptide Met-Tyr-Ser during the r efolding process. The enzyme contains 1.91 +/- 0.08 zinc atoms/mol of protein and retains full activity when stored several months at 4 degr ees C. It hydrolyzes the fluorescent substrate Dns-PLALWAR at the Ala- Leu bond with a k(cat) 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 pr oduced by activation of the Chinese hamster ovary and E. coli-expresse d 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 m ature enzyme. The pH dependence of k(cat)/K-m for Dns-PLALWAR shows th at matrilysin has a broad pH optimum (5.0-9.0) and the pK(a) values ob tained are 4.3 and 9.6 at 25 degrees C. The activity is inhibited by s everal metal binding agents including 1,10-phenanthroline, OP, but not by the nonchelating isomer, 1,7-phenanthroline OP inhibits instantane ously by likely forming a transient ternary enzyme metal chelator comp lex. The zinc atom is then removed from the protein in a time-dependen t manner. In agreement with the kinetic studies, dialysis in the prese nce 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 act ivity by addition of zinc, manganese, nickel, and cobalt, respectively . Cadmium, on the other hand, forms an inactive Cd/Zn hybrid. The diff erences in the chelator accessibility properties of the two zinc sites can thus be exploited to yield metallohybrids of matrilysin.