The vectorial specificity for calcium binding to the CaATPase of sarcoplasmic reticulum is controlled by phosphorylation, not by an E-E* conformational change.
The E-E* model for calcium pumping by the CaATPase of sarcoplasmic reticulum includes two distinct conformational states of the enzyme, E and E*. Exterior Ca2+ binds only to E and interior Ca2+ binds only to E*. Therefore, it is expected that there will be competition between the binding of calcium to the unphosphorylated enzyme from the two sides of the membrane. The equilibrium concentration of cECa2, the enzyme with Ca2+ bound at the exterior site, was measured at different Ca2+ concentrations with empty sarcoplasmic reticulum vesicles (SRV) and with SRV loaded with 40 mM Ca2+ by reaction with 0.5 mM [gamma-32P]ATP plus 20 mM EGTA for 13 ms (100 mM KCl, 5 mM MgSO4, 40 mM Mops/KOH, pH 7.0, 25 degrees C). The sigmoidal dependence on free exterior calcium concentration of the concentration of cECa2, measured as [32P]phosphoenzyme, is identical with empty and loaded SRV, within experimental error. The value of K0.5 is 2.8 microM, and the Hill coefficient is 2. This result shows that there is no competition between binding of Ca2+ to the outside and the inside of the membrane. This is consistent with a model in which the vectorial specificity for calcium binding is controlled by the chemical state of the enzyme, rather than a simple conformational change. It is concluded that there are not two interconverting forms of the free enzyme, E and E*, instead the vectorial specificity for binding and dissociation of Ca2+ is determined by the state of phosphorylation of the CaATPase.
Myung
J
J
Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254.
Jencks
W P
WP
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013379:Substrate Specificity
D000252:Calcium-Transporting ATPases; D002118:Calcium