The study characterized the biochemical properties of human collagenase-3 (MMP-13), a novel matrix metalloproteinase isolated from a breast tumor cDNA library. Recombinant human procollagenase-3 was expressed and purified, and its activation mechanism, substrate specificity, and inhibitor interaction were investigated. The purified procollagenase-3 was glycosylated and had a molecular mass of 60,000, with an N-terminal sequence consistent with the predicted sequence. Activation of procollagenase-3 by p-aminophenylmercuric acetate or streptomycin resulted in an intermediate form with a molecular mass of 50,000, which further processed to the active enzyme with a molecular mass of 48,000. The enzyme preferentially hydrolyzed soluble type II collagen, while being less efficient at cleaving type I or III collagen. It also efficiently hydrolyzed gelatin and synthetic peptide substrates, similar to gelatinases A and B. Active collagenase-3 was inhibited by tissue inhibitors of metalloproteinases (TIMPs) in a 1:1 stoichiometric fashion. These findings suggest that collagenase-3 plays a significant role in the turnover of connective tissue matrix constituents.The study characterized the biochemical properties of human collagenase-3 (MMP-13), a novel matrix metalloproteinase isolated from a breast tumor cDNA library. Recombinant human procollagenase-3 was expressed and purified, and its activation mechanism, substrate specificity, and inhibitor interaction were investigated. The purified procollagenase-3 was glycosylated and had a molecular mass of 60,000, with an N-terminal sequence consistent with the predicted sequence. Activation of procollagenase-3 by p-aminophenylmercuric acetate or streptomycin resulted in an intermediate form with a molecular mass of 50,000, which further processed to the active enzyme with a molecular mass of 48,000. The enzyme preferentially hydrolyzed soluble type II collagen, while being less efficient at cleaving type I or III collagen. It also efficiently hydrolyzed gelatin and synthetic peptide substrates, similar to gelatinases A and B. Active collagenase-3 was inhibited by tissue inhibitors of metalloproteinases (TIMPs) in a 1:1 stoichiometric fashion. These findings suggest that collagenase-3 plays a significant role in the turnover of connective tissue matrix constituents.