COOPERATIVE BINDING OF CA2-DICHROISM, FLUORESCENCE, AND CATALYTIC ACTIVITY( TO HUMAN INTERSTITIAL COLLAGENASE ASSESSED BY CIRCULAR)

Dissociation of Ca2+ from human interstitial collagenase induced eithe r by chelation with EGTA or by dilution resulted in loss of enzyme act ivity, a red shifted emission maximum from 334 to 340 nm and quenching of protein fluorescence by 10% at 340 nm. Circular dichroism indicate d 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 c atalytic activity on [Ca2+] was also cooperative (Hill coefficient, 1. 7-2.0; midpoint [Ca2+], 0.2 mM). The Ca2+-reconstituted protein was in distinguishable from the untreated enzyme by activity and fluorescence measurements. These results demonstrate that removal of Ca2+ from ful l-length collagenase generates a catalytically incompetent, partially unfolded state with native secondary structure but altered tertiary st ructure characterized by exposure of at least one tryptophyl residue t o a more polar environment.