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 pK(alpha) of 8.8 was involved in this process. The NH2-terminal analyses showed that trypsin- and stromelysin-activated enzymes had the same NH2 termini (Phe(88)), but 4-aminophenylmercuric acetate- and chymotrypsin-activated enzymes had Met(75) and Gln(89) or Glu(92) 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 Phe(88) is responsible for the Ca2+-independent activity of trypsin- and stromelysin-activated gelatinase. Replacing Asp(432) 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 K-m for the synthetic substrate but decreased the k(cat) and increased the half-maximal Ca2+ concentration required for enzyme activity (K-Ca) by severalfold, The effects on k(cat) and K-Ca depended on both charge and size of the side chains of the substituted amino acids, The decrease in k(cat) correlated well with the increase in K-Ca of the mutants, The orders of decrease in k(cat) and increase in cat K-Ca were wild type greater than or equal to D432E > D432N > D432G > D432K and wild type less than or equal to D432E < D432N < D432G < D432K, respectively, These data suggest that in trypsin- or stromelysin-activated enzyme, the NH2-terminal Phe(88) forms a salt linkage with Asp(432), rendering the enzyme Ca2+ independent, Ca2+ affects catalytic activity of the 4-aminophenylmercuric acetate- and chymotrypsin-activated enzymes by substituting for the salt linkage and interacting with Asp(432), This interaction generates a similar, if not identical, conformational change to that generated by the salt linkage in the protein, leading to catalysis.