Metal ion stabilization of the conformation of a recombinant 19‐kDa catalytic fragment of human fibroblast collagenase
作者:
Cynthia L. Lowry,
Gerard McGeehan,
Harry Le Vine,
期刊:
Proteins: Structure, Function, and Bioinformatics
(WILEY Available online 1992)
卷期:
Volume 12,
issue 1
页码: 42-48
ISSN:0887-3585
年代: 1992
DOI:10.1002/prot.340120106
出版商: Wiley Subscription Services, Inc., A Wiley Company
关键词: calcium;zinc;tryptophan fluorescence;denaturation
数据来源: WILEY
摘要:
AbstractA recombinant 19‐kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified fromE. coliinclusion 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∼60 μM, 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 stabili
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