Enzyme phosphorylation with inorganic phosphate causes Ca2+ dissociation from sarcoplasmic reticulum adenosinetriphosphatase.
Sarcoplasmic reticulum ATPase is phosphorylated by ATP in the presence of calcium, with a consequent reduction of the affinity of the binding sites for calcium and dissociation of the divalent cation from the enzyme. ATPase phosphorylation with Pi, on the other hand, requires prior removal of calcium from the enzyme, indicating that the energy requirement for phosphorylation of the enzyme-calcium complex can be met by ATP but not by Pi. We find that when the energy yield of the Pi reaction with the enzyme is increased by the addition of dimethyl sulfoxide to the medium, ATPase phosphorylation with Pi occurs even in the presence of calcium, and the binding sites undergo a reduction in affinity with consequent dissociation of Ca2+ from the enzyme, in analogy to the effect of ATP. It is thereby demonstrated experimentally that an essential step in the coupling of catalytic and transport activities is an interdependence and mutual ligand exclusion of the phosphorylation and calcium sites, in which ATP does not play a direct role. An important difference between the effects of ATP and Pi is that the former produces dissociation of Ca2+ inside the vesicles as the result of advancement of the catalytic cycle in the forward direction, while Pi produces dissociation of calcium into the outer medium as a consequence of equilibration of enzyme states producing a shift in the reverse direction of the enzyme cycle. These observations demonstrate how equilibration of intermediate enzyme states determines extent and direction of overall reaction flow.(ABSTRACT TRUNCATED AT 250 WORDS)
de Meis
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Inesi
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000648:ultrastructure
D010710:Phosphates; D000252:Calcium-Transporting ATPases; D002118:Calcium