Cooperative binding of Ca2+ to human interstitial collagenase assessed by circular dichroism, fluorescence, and catalytic activity.
Dissociation of Ca2+ from human interstitial collagenase induced either by chelation with EGTA or by dilution resulted in loss of enzyme activity, a red shifted emission maximum from 334 to 340 nm and quenching of protein fluorescence by 10% at 340 nm. Circular dichroism indicated that secondary structure was unaffected by EGTA. Ca2+ binding to the EGTA-treated enzyme as assessed by fluorescence was cooperative (Hill coefficient, 2.9; 50% saturation at 0.4 mM Ca2+). The dependence of catalytic activity on [Ca2+] was also cooperative (Hill coefficient, 1.7-2.0; midpoint [Ca2+], 0.2 mM). The Ca2+-reconstituted protein was indistinguishable from the untreated enzyme by activity and fluorescence measurements. These results demonstrate that removal of Ca2+ from full-length collagenase generates a catalytically incompetent, partially unfolded state with native secondary structure but altered tertiary structure characterized by exposure of at least one tryptophyl residue to a more polar environment.
Zhang
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Department of Biochemistry, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA.
Dean
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Gray
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RD
D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D002942:Circular Dichroism; D017364:Collagenases / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D017364:Collagenases; D002118:Calcium