Phosphorylation of (Ca2+)-adenosinetriphosphatase by inorganic phosphate: van't Hoff analysis of enthalpy changes

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Abstract
The Mg2+-dependent phosphorylation of [rabbit muscle] sarcoplasmic reticulum (ca2+)-ATPase by Pi (Mg2+ + Pi + E .dblarw. Mg.cntdot.E-P) was studied in purified leaky ATPase vesicles as a function of temperature (20-30.degree. C). A bireactant scheme was used to determine equilibrium constants, and the corresponding enthalpy changes (.DELTA.H.degree.vh) were determined by van''t Hoff analysis. At all temperatures, the binding of Pi and Mg2+ to the enzyme was synergistic. The equilibrium constants showed only a modest temperature dependence, with .DELTA.H.degree.vh varying from 3 to 13 kcal/mol. The .DELTA.H.degree.vh for Mg2+ binding to the unoccupied enzyme was 3 .+-. 2 kcal/mol. These data contrast with a recent calorimetric study under comparable conditions which reported no significant binding synergism and a .DELTA.H.degree.cal for Mg2+ binding of -76 kcal/mol. A possible reason for the discrepancy between calorimetric and van''t Hoff enthalpy determinations is given. The overall reaction was accompanied by a positive entropy change.