Cation binding by peptide substrates of collagenase may govern enzyme's specificity

Vertebrate collagenase displays high specificity in its cleavage of the collagen substrate by cleaving only one of many available Gly-Leu/Ile bonds in collagen. To understand the structural basis of this specificity, we studied the model peptides: Ac-PLG-s-LLG-O-ethyl ester (I), DnpPLGLWA(d-Arg)-NH2 (H), AcGPEGLRVG-O-ethyl ester (III) and SuccGPLGP-O-amidomethylcoumaryl ester (IV). Peptides I and II represent collagenase cleavage sequences in collagen, peptide IÜ is a mimic for the cleavage site in a2-macroglobulin and peptide IV represents a nonsubstrate model. Kinetic assays showed that peptides I, II and HI were substrates of the enzyme while peptide IV was not. CD spectral data on the peptides showed that the peptides assume ordered structures in water and trifluoroethanol. In the latter solvent, peptides I and III bound Ca²⁺ and Zn²⁺ while peptide II bound Ca²⁺ but not Zn²⁺. Peptide IV did not bind either cation in this solvent. Our results suggest that the collagenase cleavage segments in collagen and non-collagen substrates of collagenase could interact with Ca²⁺ and the enzyme to form a ternary complex. This, in turn, would imply a cofactor role for Ca²⁺ in collagenase action in addition to the solely structural role ascribed so far to this cation.