The article describes the biochemical characterization of human collagenase-3 (MMP-13), a novel matrix metalloproteinase. The cDNA for MMP-13 was isolated from a breast tumor library, and its potential role in tumor progression was proposed. The enzyme was expressed and purified, and its biochemical properties were analyzed. The purified procollagenase-3 was glycosylated and had a molecular mass of 60,000. It was activated by APMA or stromelysin, leading to the formation of an intermediate form (50,000 Da) and the final active enzyme (48,000 Da). Trypsin activation generated a Tyr85 N terminus but resulted in the loss of the C-terminal domain, reducing collagenolytic activity. The enzyme preferentially hydrolyzed soluble type II collagen, while being less efficient at cleaving type I or III collagen. It showed comparable efficiency to fibroblast and neutrophil collagenases in cleaving fibrillar type I collagen. Collagenase-3 also efficiently hydrolyzed gelatin and synthetic peptide substrates, similar to gelatinases. It was inhibited by TIMP-1, TIMP-2, and TIMP-3 in a 1:1 stoichiometric fashion. The study highlights the role of collagenase-3 in connective tissue turnover and its potential significance in tumor progression. The enzyme is a member of the matrix metalloproteinase family, which includes collagenases, stromelysins, and gelatinases. The study provides insights into the activation mechanisms, substrate specificity, and inhibitor interactions of collagenase-3, emphasizing its importance in matrix remodeling and disease processes.The article describes the biochemical characterization of human collagenase-3 (MMP-13), a novel matrix metalloproteinase. The cDNA for MMP-13 was isolated from a breast tumor library, and its potential role in tumor progression was proposed. The enzyme was expressed and purified, and its biochemical properties were analyzed. The purified procollagenase-3 was glycosylated and had a molecular mass of 60,000. It was activated by APMA or stromelysin, leading to the formation of an intermediate form (50,000 Da) and the final active enzyme (48,000 Da). Trypsin activation generated a Tyr85 N terminus but resulted in the loss of the C-terminal domain, reducing collagenolytic activity. The enzyme preferentially hydrolyzed soluble type II collagen, while being less efficient at cleaving type I or III collagen. It showed comparable efficiency to fibroblast and neutrophil collagenases in cleaving fibrillar type I collagen. Collagenase-3 also efficiently hydrolyzed gelatin and synthetic peptide substrates, similar to gelatinases. It was inhibited by TIMP-1, TIMP-2, and TIMP-3 in a 1:1 stoichiometric fashion. The study highlights the role of collagenase-3 in connective tissue turnover and its potential significance in tumor progression. The enzyme is a member of the matrix metalloproteinase family, which includes collagenases, stromelysins, and gelatinases. The study provides insights into the activation mechanisms, substrate specificity, and inhibitor interactions of collagenase-3, emphasizing its importance in matrix remodeling and disease processes.