Slow dissociation of ATP from the calcium ATPase.

The acyl-phosphate intermediate of the sarcoplasmic reticulum calcium ATPase reaction, formed in a brief incubation of vesicular enzyme with 5 microM [gamma-32P]ATP and calcium, reacts biphasically with added ADP (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4). Both the burst size and the rate constant for the slow phase increase with increasing ADP concentration in the way that is expected if the burst represents very rapid formation of an equilibrium amount of enzyme-bound ATP and the slow phase represents rate-limiting dissociation of ATP. Also consistent with this interpretation are the slow labeling of phosphoenzyme under conditions in which unlabeled ATP must dissociate first and the observation of a burst of ATP formation on ADP addition to phosphoenzyme. Values of the equilibrium constants for ADP dissociation from phosphoenzyme (0.75 mM), for ATP formation on the enzyme (2.3), and for the ATP dissociation rate constant (37 s-1) were obtained from a quantitative analysis of the data.