DISSOCIATION OF CALCIUM FROM THE PHOSPHORYLATED CALCIUM-TRANSPORTING ADENOSINE-TRIPHOSPHATASE OF SARCOPLASMIC-RETICULUM - KINETIC EQUIVALENCE OF THE CALCIUM-IONS BOUND TO THE PHOSPHORYLATED ENZYME

The internalization of Ca-45 by the calcium-transporting ATPase into sarcoplasmic reticulum vesicles from rabbit muscle was measured during a single turnover of the enzyme by using a quench of 7 mM ADP and EGTA (25-degrees-C, 5 mM MgCl2, 100 mM KCl, 40 mM MOPS-Tris, pH 7.0). Intact vesicles containing either 10-20-mu-M or 20 mM Ca2+ were preincubated with Ca-45 for approximately 20 s and then mixed with 0.20-0.25 mM ATP and excess EGTA to give 70% phosphorylation of E(tot) with the rate constant k = 300 s-1. The two Ca-45 ions bound to the phosphoenzyme (EP) become insensitive to the quench with ADP as they are internalized in a first-order reaction with a rate constant of k = approximately 30 s-1. The first and second Ca2+ ions that bind to the free enzyme were selectively labeled by mixing the enzyme and Ca-45 with excess Ca-40, or by mixing the enzyme and Ca-40 with Ca-45, for 50 ms prior to the addition of ATP and EGTA. The internalization of each ion into loaded or empty vesicles follows first-order kinetics with k = approximately 30 s-1; there is no indication of biphasic kinetics or an induction period for the internalization of either Ca2+ ion. The presence of 20 mM Ca2+ inside the vesicles has no effect on the kinetics or the extent of internalization of either or both of the individual ions. The Ca2+ ions bound to the phosphoenzyme are kinetically equivalent. A first-order reaction for the internalization of the individual Ca2+ ions is consistent with a rate-limiting conformational change of the phosphoenzyme with k(c) = 30 s-1, followed by rapid dissociation of the Ca2+ ions from separate independent binding sites on E approximately P.Ca2; lumenal calcium does not inhibit the dissociation of calcium from these sites. Alternatively, the Ca2+ ions may dissociate sequentially from E approximately P.Ca2 following a rate-limiting conformational change. However, the order of dissociation of the individual ions can not be distinguished. An ordered-sequential mechanism for dissociation requires that the ions dissociate much faster (k greater-than-or-equal-to 10(5) s-1) than the forward and reverse reactions for the conformational change (k(-c) = approximately 3000 s-1). Finally, the Ca2+ ions may exchange their positions rapidly on the phosphoenzyme (k(mix) greater-than-or-equal-to 10(5) s-1) before dissociating. A Hill slope of n(H) = 1.0-1.2, with K0.5 = 0.8-0.9 mM, for the inhibition of turnover by binding of Ca2+ to the low-affinity transport sites of the phosphoenzyme was obtained from rate measurements at six different concentrations of Mg2+.