Analysis of the role of the COOH-terminal domain in the activation, proteolytic activity, and tissue inhibitor of metalloproteinase interactions of gelatinase B.

Recombinant human progelatinase B and a COOH terminally truncated version, pro-delta426-688 gelatinase B have been prepared from a myeloma cell expression system. Both proenzymes could be processed to active forms by stromelysin-1 to give an NH2 terminus of Phe88, or by treatment with 4-aminophenylmercuric acetate resulting in an NH2-terminal Met75. The kinetics of activation using either treatment was not affected by removal of the enzyme COOH-terminal domain. The specific activities of both gelatinase B and delta426-688 gelatinase B, activated using either method, were found to be similar using either a quenched fluorescent peptide or gelatin as the substrate. Fibroblast monolayers were shown to mediate processing of both progelatinases at similar rates in the presence of either plasminogen or prostromelysin-1. Active wild-type gelatinase B was inhibited by tissue inhibitor of metalloproteinase (TIMP) -1 at a much faster rate than TIMP-2. COOH-terminal truncation of either enzyme or inhibitor gave a marked reduction in the rate constant for TIMP-1 inhibition but had no effect on the rate of TIMP-2 binding. It can be concluded that the COOH-terminal domain of progelatinase B is not involved in autolytic or cellular activation and does not affect the catalytic activity of the enzyme. However, COOH-terminal domain interactions between active gelatinase B and TIMP-1 significantly enhance the rate of complex formation.