Metal ion stabilization of the conformation of a recombinant 19-kDa catalytic fragment of human fibroblast collagenase.
A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 approximately 60 microM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium.
Lowry
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Glaxo Research Laboratories, Research Triangle Park, North Carolina 27709.
McGeehan
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LeVine
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D002942:Circular Dichroism; D004795:Enzyme Stability; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D008670:Metals / Q000737:chemistry*; D003012:Microbial Collagenase / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D008670:Metals; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003012:Microbial Collagenase