Reaction mechanism of Ca2+-dependent ATP hydrolysis by skeletal muscle sarcoplasmic reticulum in the absence of added alkali metal salts. III. Sequential occurrence of ADP-sensitive and ADP-insensitive phosphoenzymes.

The characteristics of the phosphoenzyme intermediates formed in the absence of added alkali metal salts by a partially purified adenosine triphosphatase preparation of skeletal muscle sarcoplasmic reticulum were examined. Two types of acid-stable phosphoenzyme could be demonstrated during the initial transient phase of Ca2+-dependent ATP hydrolysis in low free Mg"+ and Ca2+ concentrations at 0" and pH 6.8. One of these phosphoenzymes, designated E,P, was ADP-sensitive as it could donate its phosphate group to added ADP to form ATP. The other, designated E2P, was ADP-insensitive as it did not donate its phosphate group to added ADP. Evidence is presented that during ATP hydrolysis under these conditions, E,P was formed first and then converted to EZP. This conversion was accelerated by free Mg2+, an effect that was antagonized by free Ca2+. This action of Ca2+ appeared to reflect its ability to accelerate the reverse reaction of the E,P + E,P conversion. The free Mg*+ concentration that produced half-maximal acceleration of the E,P -+ E2P conversion in 20 FM free Ca2+ was approximately 20 PM. The free CaZ+ concentration that produced half-maximal acceleration of the E,P -+ E,P conversion was 0.6 to 1.0 mu in 1 to 2 mu Mg'+, and was affected only slightly by changing free Mg*+ concentration. Furthermore, a significant conversion of E,P -+ E,P could not be demonstrated in low free Ca2+ concentrations (-20 PM).