We are investigating the in vitro mechanism of activation of the matrix metalloproteinase Gelatinase A. Activation of the enzyme, normally secreted in a latent form, is a critical control step in determining the amount of matrix degradation contributed by this enzyme in vivo. By understanding, in detail, the in vitro mechanism of activation, we hope to develop targets for therapeutic intervention. From previous studies, we know that this enzyme is secreted as a pro-enzyme, often in complex with its specific inhibitor, tissue inhibitor of metalloproteinases-2, TIMP-2. Latency of the pro-enzyme is maintained by preservation of a covalent bond between a cysteine sulfhydryl and the active site zinc atom. Physical disruption (denaturation) or chemical cleavage (organomercurial) of this bond result in a series of events which end in loss of 80 amino acids from the amino-terminus and development of proteolytic activity. In vivo, activation occurs by a cell-surface associated mechanism that gives the same result as in vitro activation. Therefore, a clear understanding of the order and rate of events in vitro will also allow better understanding of the in vivo mechanism. In parallel with the kinetic analysis of the activation mechanism, we also seek to understand how inhibitors, including TIMPs, TIMP fragments or analogues and peptide inhibitors based on the activation locus inhibit the processes of activation and catalysis.
