Phosphorylation of calcium adenosinetriphosphatase by inorganic phosphate: van't Hoff analysis of enthalpy changes

The magnesium-dependent phosphorylation of sarcoplasmic reticulum (Ca2+)-AtPase by inorganic phosphate (Mg2+ + Pi + E = Mg.E-P) was studied in purified leaky AtPase vesicles as a function of temperature (20-30 degrees C). A bireactant scheme was used to determine equilibrium constants, and the corresponding enthalpy changes ( delta H degrees vh) were determined by van't Hoff analysis. At all temperatures, the binding of Pi and Mg(2+) to the enzyme was synergistic. The equilibrium constants showed only a modest temperature dependence, with delta H degrees vh varying from 3 to 13 kcal/mol. In particular, the delta H degrees vh Mg(2+) binding to the unoccupied enzyme was 3 +/ 2 kcal/mol. These data contrast with a recent calorimetric study under comparable conditions (Epstein, M., Kuriki, Y., Biltonen, R., & Racker, E. (1980) Biochemistry 17, 5564) which reported no significant binding synergism and a delta H degrees cal for Mg(2+) binding of -76 kcal/mol. A possible reason for the discrepancy between calorimetric and van't Hoff enthalpy determinations is given. In agreement with other previous work, the overall reaction was found to be accompanied by a positive entropy change.