The thermodynamics of calcium binding to thermolysin.

Calcium binding isotherms were determined for thermolysin in the range pH 5.6-10.5, and from 5 to 45 degrees C. An extensive statistical analysis of the binding data suggests that at least two of the four binding sites bind Ca2+ with complete positive cooperativity and independently of the other two. Nonlinear regression analysis of the binding data was used to calculate cooperative (K1) and independent (K2) binding constants for the four calcium sites. Thermodynamic parameters obtained from a van't Hoff analysis indicate that calcium binding to both cooperative and independent sites is an entropy-driven process. At pH 7.0, delta H1 = 90.4 kJ/mol; delta H2 = 97.5 kJ/mol; delta S1 = 456 J K-1 mol-1; delta S2 = 262 J K-1 mol-1. These results are compared to those obtained for other calcium-binding proteins. An analysis of the pH dependence of the calcium binding constants indicates that the binding of four protons at the cooperative site and one to two protons at the independent sites, modulates the calcium affinity. This confirms an earlier structural assignment of the double-site as the locus of the two cooperatively binding Ca2+. Calcium binding to thermolysin is enhanced in the presence of an active site directed inhibitor, suggesting that there may be positive cooperativity between substrate and calcium binding.