Purification, inhibition and mechanistic studies of Clostridium histolyticum and human neutrophil collagenases

The specific collagenase stored in the granules of human neutrophils has been purified by a simple, reproducible method. A sensitive, accurate and convenient assay for tissue collagenases has been developed to aid the purification. This assay, which is based on the hydrolysis of [3H]acetylated rat tail tendon type I collagen, has also enabled the inhibition of this enzyme to be studied quantitatively. The purified enzyme has very high specific activity towards type I collagen and is devoid of other contaminating proteolytic activities. It is a zinc metalloproteinase that requires calcium ions for activity. In addition, it contains essential lysine, tyrosine and carboxyl residues within the active site. The types of compounds that have been found to be good inhibitors for other zinc metalloproteinases also inhibit both the human neutrophil and the class I and class II Clostridial collageneases. The Clostridial enzymes are strongly inhibited by phosphonoamidates and ketone analogs that contain collagen-like sequences. This pattern of inhibition suggests a remarkable similarity between the mechanism of action of these collagenases and other well studied zinc metalloproteinases. The inhibition data also suggest the possible use of these inhibitors as pharmaceuticals and as affinity ligands for the purification of the collagenases. In fact, the use of the ketone analogs to purify the class I and class II Clostridial collagenases from crude mixtures has been successfully demonstrated