Mechanism of activation of human neutrophil gelatinase B. Discriminating between the role of Ca2+ in activation and catalysis.

Gelatinase B is a Zn(2+)- and Ca(2+)-dependent endopeptidase that is secreted from cells as an inactive proenzyme. The enzyme can be activated in vitro by organomercurial compounds and by trypsin. The role of Ca2+ in autoproteolytic processing initiated by 4-aminophenylmercuric acetate and trypsin and in catalytic activity of the activated enzyme was investigated by zymography and by kinetic analysis. Treatment of unglycosylated 57.5-kDa pro-gelatinase B with 4-aminophenylmercuric acetate (1 mM) in the absence of Ca2+ generated a 49-kDa inactive intermediate (E'), whereas a 41.5-kDa active species (E") was generated in the presence of Ca2+ (5 mM). Upon addition of Ca2+ to the reaction mixture of Ca(2+)-depleted E' or E" at 37 degrees C, E' showed a lag period in generation of the product as a function of time, but E" presented an immediate activity. The appearance of enzymatic activity of E' correlated with the generation of the E" species. NH2-terminal sequence analyses showed that E' and E" had the same NH2 termini, i.e. Met-75, suggesting that Ca(2+)-dependent removal of COOH terminus of E' is required for activation of the enzyme. Treatment of pro-gelatinase B with trypsin in the absence of Ca2+, led to degradation of the enzyme. In the presence of Ca2+, trypsin processed the pro-enzyme to a 40-kDa active species. In contrast to E", this active species did not require Ca2+ for activity. The Ca2+ dependence of E" activity was also abolished by treatment of the enzyme with trypsin. NH2-terminal sequence analysis indicated that amino acid residues 75-87 had been removed from the NH2 terminus of E" by trypsin, suggesting that these residues are responsible for the Ca(2+)-dependent activity of the enzyme. Removal of Ca2+ and catalytic Zn2+ inhibited the activities of both E" and trypsin-treated E". In the absence of Ca2+, either Zn2+, Co2+, Mn2+, or Cd2+ was able to restore the activity of trypsin-treated E". None of the divalent cations tested however, was able to stimulate the activity of E" in the absence of Ca2+. These experiments further suggest that binding of Ca2+ to E" or removal of the NH2-terminal residues of the enzyme by trypsin induces a conformational change in the protein and makes the active site of the enzyme accessible to various metal ions rendering the enzyme active.