Site-directed mutagenesis of the active site glutamate in human matrilysin: Investigation of its role in catalysis

Glu-198 of human matrilysin is a conserved residue in the matrix metalloproteinases and is considered to play an important role in catalysis by acting as a general base catalyst toward the zinc-bound water molecule, on the basis of mechanistic proposals for other zinc proteases. In the present study, Glu-198 is mutated into Asp, cys, Gln, and Ala, and the zinc binding properties, kinetic parameters and pH dependence of each mutant are determined in order to examine the role of Glu-198 in catalysis. The mutations chosen either modify (C and D) or eliminate (A and Q) the general base properties of residue-198. All the mutants bind 2 mol of zinc per mol of enzyme, indicating that Glu-198 is not crucial to the binding of the catalytic zinc to the enzyme. The value of k(cat)/K-m for the E198C mutant is only 4-fold lower than that of wild-type enzyme at the pH optimum of 7.5, while that for the E198C mutant is decreased by 160-fold. The E198Q and E198A enzymes containing the mutations that have eliminated the nucleophilis and acid/base properties of the residue are still active, having lower k(cat)/K-m values of 590- and 1900-fold, respectively, The decrease in activity of all the mutants is essentially due to a decrease in k(cat). The k(cat)/K-m values of the mutants as a function of pH display broad bell-shaped curves that are similar to the wild-type enzyme. The acidic pK(a) value is not greatly affected by the change in the chemical that the ionization of Glu-198 is not responsible for the acidic pK(a). Ionization of the zinc-bound water may be responsible for this pK(a) since the three His ligands and the scaffoling of the matrilysin catalytic zinc site are different form that observed in carboxypeptidase A and would predict a lower pK(a) for the metal-bound water. If the zinc-bound water is the nucleophile in the reaction, the role of Glu-198 in catalysis may be to stabilize the transition state or act a a general acid catalyst after the rate-determining step.