Binding of two Sr2+ ions changes the chemical specificities for phosphorylation of the sarcoplasmic reticulum calcium ATPase through a stepwise mechanism.
The sequential binding of Sr2+ and Ca2+ to the cytoplasmic transport sites of the sarcoplasmic reticulum calcium ATPase allows the formation of two different mixed complexes: cE.Sr.Ca, with Sr2+ bound to the "inner" site and Ca2+ bound to the "outer" site, and cE. Ca.Sr, with Ca2+ bound to the inner site and Sr2+ bound to the outer site (pH 7.0, 25 degrees C, 10 mM MgCl2, 100 mM KCl). Both cE.Sr.45Ca and cE.45Ca.Sr react with ATP to internalize one 45Ca/phosphoenzyme. The value of K0.5 = 83 microM Sr2+ for activation of the enzyme for phosphorylation by ATP is much larger than K0.5 = 28 microM Sr2+ for inhibition of phosphoenzyme formation from inorganic phosphate (eta H = 1.0-1.3). These results are consistent with the sequential binding of two strontium ions with negative cooperativity and dissociation constants of KSr1 = 35 microM and KSr2 = 55 microM. The species cE.Sr2 and cE.Ca2 react rapidly with ATP but not inorganic phosphate. However, enzyme with one strontium bound, cE.Sr, does not react with either inorganic phosphate or ATP. Therefore, the conformational changes in the enzyme that alter the chemical specificity for phosphorylation by ATP and by inorganic phosphate are different. This requires the existence of at least three forms of the unphosphorylated enzyme with three different chemical specificities for catalysis.
Fujimori
T
T
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Jencks
W P
WP
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002413:Cations, Divalent; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013324:Strontium / Q000378:metabolism*
D002413:Cations, Divalent; D010710:Phosphates; D000252:Calcium-Transporting ATPases; D002118:Calcium; D013324:Strontium