Mechanism of Ca2+-dependent activity of human neutrophil gelatinase B

Progelatinase B can be activated in vitro by organomercurial compounds and by proteolytic enzymes such as trypsin, chymotrypsin, and stromelysin. Activation of the proenzyme by either 4-aminophenylmercuric acetate or chymotrypsin yielded proteins that absolutely required Ca2+ for activity, regardless of the pH of the reaction mixture. The trypsin- and stromelysin-activated gelatinases, on the other hand, did not require Ca2+ for activity at pH 7.5, but the activity of the trypsin-activated enzyme became Ca2+ dependent as the pH increased. The pH study revealed that an amino acid residue with an apparent pKa of 8.8 was involved in this process. The NH2-terminal analyses showed that trypsin- and stromelysin-activated enzymes had the same NH2 termini (Phe88), but 4-aminophenylmercuric acetate- and chymotrypsin-activated enzymes had Met75 and Gln89 or Glu92 as the NH2-terminal amino acid, respectively. These data, in conjunction with the x-ray crystal structure of collagenase, suggest that a salt linkage involving Phe88 is responsible for the Ca2+-independent activity of trypsin- and stromelysin-activated gelatinase. Replacing Asp432 in progelatinase with either Glu, Asn, Gly, or Lys resulted in the proteins that, upon activation by trypsin, required Ca2+ for activity. These substitutions did not significantly affect Km for the synthetic substrate but decreased the kcat and increased the half-maximal Ca2+ concentration required for enzyme activity (KCa) by severalfold. The effects on kcat and KCa depended on both charge and size of the side chains of the substituted amino acids. The decrease in kcat correlated well with the increase in KCa of the mutants. The orders of decrease in kcat and increase in KCa were wild type >/= D432E > D432N > D432G > D432K and wild type