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 °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 × 10^-5 M at pH 7.5, 37 °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 k_cat/K_m for Dns-PLALWAR shows that matrilysin has a broad pH optimum (5.0−9.0) and the pK_a values obtained are 4.3 and 9.6 at 25 °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.