The sarcoplasmic reticulum Ca2+-ATPase was phosphorylated with either p-nitrophenyl phosphate (pNPP) or with ATP in the pres ence of Ca2+ under the condition that the free energy change of pNPP hydrolysis is less than that of ATP, Delta G'(pNPP) < Delta G'(ATP), or the condition that Delta G'p(NPP) approximate to Delta G'(ATP), without any additional energy input. In both cases, the pNPP-prepared phosphoenzyme synthesized ATP upon the simple addition of ADP, as did the phosphoenzyme prepared with ATP. The sensitivity of these phosphoenzymes to ADP for the synthesis of ATP was the same. In other words, there is no hysteresis of the phosphate donor to its Delta G' value in these phosphoenzymes. This result means that both phosphoenzymes by themselves must have sufficient conformational energy, independent of the Delta G' value of the phosphate donor, for the reversed synthesis of ATP, because a high-energy phosphate donor (ATP) cannot be formed from a lower-energy phosphate donor (pNPP). (c) 2006 Elsevier Inc. All rights reserved.