Characterization of Ca2+ interactions with matrix metallopeptidase-12: implications for matrix metallopeptidase regulation.
Matrix metallopeptidase-12 (MMP-12) binds three calcium ions and a zinc ion, in addition to the catalytic zinc ion. These ions are thought to have a structural role, stabilizing the active conformation of the enzyme. To characterize the importance of Ca2+ binding for MMP-12 activity and the properties of the different Ca2+ sites, the activity as a function of [Ca2+] and the effect of pH was investigated. The enzymatic activity was directly correlated to calcium binding and a Langmuir isotherm for three binding sites described the activity as a function of [Ca2+]. The affinities for two of the binding sites were quantified at several pH values. At pH 7.5, the KD was 0.1 mM for the high-affinity binding site, 5 mM for the intermediate-affinity binding site and >100 mM for the low-affinity binding site. For all three sites, the affinity for calcium decreased with reduced pH, in accordance with the loss of interactions upon protonation of the calcium-co-ordinating aspartate and glutamate carboxylates at acidic pH. The pKa values of the calcium binding sites with the highest and intermediate affinities were determined to be 4.3 and 6.5 respectively. Optimal pH for catalysis was above 7.5. The low-, intermediate- and high-affinity binding sites were assigned on the basis of analysis of three-dimensional-structures of MMP-12. The strong correlation between MMP-12 activity and calcium binding for the physiologically relevant [Ca2+] and pH ranges studied suggest that Ca2+ may be involved in controlling the activity of MMP-12.
Gossas
Thomas
T
Department of Biochemistry and Organic Chemistry, Uppsala University, Uppsala, Sweden.
Danielson
U Helena
UH
D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053508:Matrix Metalloproteinase 12; D008666:Metalloendopeptidases / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation
D008666:Metalloendopeptidases; C507122:MMP12 protein, human; D053508:Matrix Metalloproteinase 12; D002118:Calcium