Binding of Ca2+ to the calcium adenosinetriphosphatase of sarcoplasmic reticulum

The binding of Ca/sup 2 +/ and the resulting change in catalytic specificity that allows phosphorylation of the calcium ATPase of sarcoplasmic reticulum by ATP were examined by measuring the amount of phosphoenzyme formation from (/sup 32/P)ATP, or /sup 45/Ca incorporation into vesicles, after the simultaneous addition of ATP and EGTA at different times after mixing enzyme and Ca/sup 2 +/. A burst of calcium binding in the presence of high (Ca/sup 2 +/) gives /approximately/ 12% phosphorylation and internalization of two Ca/sup 2 +/ at very short times after the addition of Ca/sup 2 +/ with this assay. This shows that calcium binding sites are available on the cytoplasmic-facing side of the free enzyme. Calcium binding to these sites induces the formation of /sup c/E/times/Ca/sub 2/, the stable high-affinity form of the enzyme, with k = 40 s/sup -1/ at saturating (Ca/sup 2 +/) and a half-maximal rate at approximately 20 ..mu..M Ca/sup 2 +/. The formation of /sup c/E/times/Ca/sub 2/ through a high-affinity pathway is described. The rate of exchange of /sup 45/Ca into the inner position of /sup c/E/times/Ca/sub 2/ shows and induction period and is not faster than the approach to equilibrium starting with E andmore » /sup 45/Ca. The dissociation of /sup 45/Ca from the inner position of /sup c/E/times//sup 45/Ca/times/Ca in the presence of 3.2 ..mu..M Ca/sup 2 +/ occurs with a rate constant of 7 s/sup -1/. These results are inconsistent with a slow conformational change of free E to give /sup c/E, followed by rapid binding-dissociation of Ca/sup 2 +/.« less