The purified collagenase from tadpole (Rana catesbiana) back skin was studied with respect to its activation energy using soluble and fibrillar type I collagen, as well as a synthetic peptide substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. The activation energy appeared to be independent of the nature of the substrate, ranging between 28 and 35 kcal/mol. The peptide was cleaved at the Gly-Ile bond and proved to be a poor substrate (kcat/Km, 1.21 h-1 microM-1) when compared with native type I collagen in solution (kcat/Km, 40.6 h-1 microM-1), consistent with the enzyme's low activity versus gelatin [T. A. Bicsak and E. Harper (1984) J. Biol. Chem. 259, 13145]. The amino acid composition of the collagenase was shown to be high in glycine and glutamic acid, and the preparation was shown not to be contaminated with collagen by digestion with bacterial collagenase. The enzyme was not inhibited by iodoacetic acid or 2-hydroxy-5-nitrobenzyl bromide, suggesting the lack of essential cysteinyl and tryptophanyl residues, but was inhibited by micromolar concentrations of ZnCl2, consistent with the presence of essential histidine(s). Ethoxyformic anhydride irreversibly inhibited the collagenase suggesting the presence of essential lysyl residues.