Cooperative Binding of Ca2+ to Human Interstitial Collagenase Assessed by Circular Dichroism, Fluorescence, and Catalytic Activity

Abstract

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. Ca 2؉ 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.

Involvement of fibrinogen specific binding in erythrocyte aggregation

“…The bound fibrinogen concentration was determined using a standard curve of OG‐Fb fluorescence. Specific binding at each OG‐Fb concentration was determined from the difference between total binding (absence of unlabeled fibrinogen) and non‐specific binding (100‐fold excess of unlabeled fibrinogen) [9]. Autofluorescence at 524 nm was less than 1% of the total fluorescence.…”

Mutations in the Catalytic Domain of Human Matrix Metalloproteinase-1 (MMP-1) That Allow for Regulated Activity through the Use of Ca2+

“…11 and references therein). The calcium molecules have also been shown to play an important role in domain stabilization and in the regulation of catalytic activity (12)(13)(14). As expected, the amino acids important for the binding of the zinc and calcium molecules within the catalytic domain are highly conserved among MMP family members (11).…”

“…As previously mentioned, calcium has been shown to play an important role in domain stabilization and tertiary structure formation (12)(13)(14). In fact, under certain conditions, including low calcium concentrations, MMP-1 and MMP-3 have been shown to undergo autolysis (13,19).…”

“…Although Ca 2ϩ does not directly participate in proteolysis, it is integral in the stabilization of the tertiary structure of the enzyme (12). In fact, circular dichroism analysis of human MMP-1 revealed that in the absence of Ca 2ϩ , the protein is in a catalytically inactive and partially unfolded state with native secondary structure but altered tertiary structure (14). Over time, in the absence of Ca 2ϩ , the enzymatic loss is irreversible, and in the case of MMP-3, it results in autocatalysis (13).…”

Cooperative binding of calcium ions modulates the tertiary structure and catalytic activity of Matrix-Metalloproteinase-9

“…Due to their relative locations on the protein surface (i.e., far removed from the active site), non-catalytic Zn 2+ and all Ca 2+ appear to exhibit structural rather than catalytic roles (Figure 1). Contrary to this expectation, several investigators noted that besides performing the structural roles [29][30][31], the binding of Ca 2+ enhances the catalytic activities of several MMPs [32][33][34], including those of MMP-2 and MMP-9 [35][36][37][38]. By employing dynamic simulation studies on MMP-2, Diaz and Suarez [35] demonstrated that both non-catalytic Zn 2+ and Ca…”