The metabolic turnover of the major protein constituents of the extracellular matrix is catalyzed by a family of enzymes called the matrix metalloproteinases (MMP). Two members of this gene family, human fibroblast collagenase (HFC) and human neutrophil collagenase (HNC), are the only human enzymes capable of hydrolyzing the fibrillar type I, II and III collagens at an appreciable rate under physiological conditions. They do so in a highly specific and characteristic manner by making a single scission across all three chains of the tropocollagen (TC) monomers at a specific, sensitive locus approximately 3/4 from the N-terminus to produce TCA and TCB fragments. This reaction, which is one of the most specific known in enzymology, is the committed step in collagen turnover. The goal of our studies is to elucidate the underlying basis for this remarkable molecular recognition system from the point of view of both the collagenase and the collagenase cleavage site in the collagens. Samples of HNC will be prepared for crystal growth experiments so that the structure of both the zymogen (pro-HNC) and active enzyme (HNC) can be elucidated by x-ray crystallography. Recombinant zymogen (r-pro-HNC) will be expressed in E. coli and isolated as a source of unglycosylated enzyme for x-ray crystallographic studies. Mutant r-pro-HNC species will be prepared with altered sequences at the autolytic activation and degradation sites to improve the stability of the enzyme at the concentrations used in crystal growth experiments. The role of the hemopexin-like domain of HFC and HNC in determining their substrate specificity will be investigated by carrying out a series of domain switching experiments between these two collagenases, as well as between them and other MMP. Information about the collagenase cleavage site in collagens will be investigated by preparing a series of model, triple helical peptide substrates by solid phase peptide synthesis. The action of HFC and HNC on models with ascending levels of resemblance to the collagenase cleavage site will be studied in order to elucidate features of this site that are critical for recognition and cleavage by human collagenases. Crystallographic studies of the model triple helical peptides whose recognition and cleavage by HFC and HNC most closely mimic that of the natural collagen substrates will be carried out to elucidate the local structure of the cleavage site.
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