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.