Mechanism of Ca2+-dependent Activity of Human Neutrophil Gelatinase B

Chun Hui Bu, Tayebeh Pourmotabbed
1996 Journal of Biological Chemistry  
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 trypsinactivated
more » ... e became Ca 2؉ dependent as the pH increased. The pH study revealed that an amino acid residue with an apparent pK 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. The abbreviations used are: MMP, matrix metalloproteinase; APMA, 4-aminophenylmercuric acetate; E a , APMA-activated gelatinase B; E t , trypsin-activated gelatinase B; WT, wild-type; Mca, (7-methoxycoumarin-4-yl) acetic acid; Dpa, 3-(2Ј,4Ј-dinitrophenyl)-L-2,3-diaminopropionic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid.
doi:10.1074/jbc.271.24.14308 pmid:8662913 fatcat:esjsfzijdjgk7g3rmlminfabw4