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The CaATPase of sarcoplasmic reticulum was reacted with [gamma-32P]ATP to form the covalent phosphoenzyme intermediate. Noncompetitive inhibition by reactive red-120 and chelation of calcium allowed us to monitor single-turnover kinetics of the phosphoenzyme reacting with water or added ADP at 0 degrees C. When ADP was added and the amount of product, [gamma-32P]ATP, formed was measured, we found that added cold ATP did not interfere with the phosphoenzyme reacting with ADP. We conclude that ATP cannot bind where ADP binds, the phosphorylated active site. This implies that when ATP at high concentrations causes an acceleration of phosphoenzyme hydrolysis, it must do so by binding to an allosteric site. Considering the monoexponential nature of product formation we observed, simple one-nucleotide-site models cannot account for the above result.

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