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

Abstract

To ascertain the molecular basis of Ca 2+-mediated activation of matrix metalloproteinase-9 (MMP-9), we determined the accessibility of tryptophan residues to externally added acrylamide as quencher in the absence and presence of the metal ion. The steady-state and time resolved fluorescence data revealed that MMP-9 possesses two classes of tryptophan residues, "exposed" and "buried" which are quenched by the collisional rate constants (k q ) of 3.2  10 ; the steady-state acrylamide quenching data could only be explained by a combination of "dynamic" and "static" quenching models. A cumulative account of these data led to the suggestion that the binding of Ca 2+ modulated the tertiary structure of the protein by decreasing the dynamic flexibility of the enzyme, which is manifested in further structuring of the enzyme's active site pocket toward facilitating catalysis. Arguments are presented that the binding of Ca 2+ at distal sites "dynamically" communicates with the active site residues of MMP-9 during catalysis.

Nuclear Localizing Peptide-Conjugated, Redox-Sensitive Polymersomes for Delivering Curcumin and Doxorubicin to Pancreatic Cancer Microtumors

“…Like other MMPs, MMP-1 was found to be confined in the inclusion bodies from where it was purified by solubilizing in buffer (50 mM Tris-HCl, pH 7.5) containing 6 M urea, 10 mM CaCl 2 , and 10 μM ZnCl 2 followed by subjecting the suspension to serial dialysis against the above buffer with decreasing concentration of urea as described by Tobwala and Srivastava. 47 The folded protein exhibited high catalytic activity and showed a predominant band on SDS−PAGE. The cloning, expression, and purification of human MMP-7 were performed as described by Ganguly et al 48 Polymersome Preparation.…”