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 Ca 2+ for activity, regardless of the pH of the reaction mixture. The trypsin- and stromelysin-activated gelatinases, on the other hand, did not require Ca 2+ for activity at pH 7.5, but the activity of the trypsin-activated enzyme became Ca 2+ dependent as the pH increased. The pH study revealed that an amino acid residue with an apparent p K a of 8.8 was involved in this process. The NH 2 -terminal analyses showed that trypsin- and stromelysin-activated enzymes had the same NH 2 termini (Phe 88 ), but 4-aminophenylmercuric acetate- and chymotrypsin-activated enzymes had Met 75 and Gln 89 or Glu 92 as the NH 2 -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 Ca 2+ -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 Ca 2+ for activity. These substitutions did not significantly affect K m for the synthetic substrate but decreased the k cat and increased the half-maximal Ca 2+ 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 K Ca were wild type ≥ D432E > D432N > D432G > D432K and wild type ≤ D432E < D432N < D432G < D432K, respectively. These data suggest that in trypsin- or stromelysin-activated enzyme, the NH 2 -terminal Phe 88 forms a salt linkage with Asp 432 , rendering the enzyme Ca 2+ independent. Ca 2+ 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.