Studies on the mechanism of action of collagenase: inhibition by cysteine and other chelating agents

Abstract Under specific conditions cysteine inhibits collagenase (Clostridiopeptidase A and B (EC 3.4.4.19)) irreversibly. Neither collagenase A or B contains cysteinyl or cystinyl residues, so that the inhibition cannot proceed either by disulfide reduction, formation, or exchange. By use of [ 35 S]cysteine, cysteine has been shown to bind to collagenase A in the ratio of about two moles to one mole of protein, and to collagenase B in the ratio of approximately one mole to one mole of protein. After cysteine binds to the enzyme, the sulfhydryl group can no longer be titrated with p ‐mercuribenzoate or 5,5′‐dithiobis‐(2‐nitrobenzoic acid). Cysteine inhibits the low‐temperature binding of collagenase to a collagen substrate (ichthyocol). Histidine inhibits the activity of collagenases A and B, but this inhibition, in contrast to that produced by cysteine, is readily reversible by dialysis against water or passage through a DEAE cellulose column. 2,3‐Dimercaptopropanol also inhibits both collagenases. The order of the inhibitor efficacy on collagenase by cysteine, histidine, imidazole and 2,3‐dimercaptopropanol appears to be consistent with the order of stability constants of these chelating agents with zinc. The suggestion made previously that zinc may be an intrinsic component of collagenase is thereby reinforced. Indeed preliminary experiments are reported in which the incorporation of 65 Zn into collagenases A and B is demonstrated.