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The vectorial specificity for calcium binding to the CaATPase of sarcoplasmic reticulum is controlled by phosphorylation, not by an E—E* conformational change
Author(s) -
Jaekyung Myung,
William P. Jencks
Publication year - 1991
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(91)80077-g
Subject(s) - endoplasmic reticulum , egta , chemistry , calcium , conformational change , enzyme , biophysics , vesicle , binding site , membrane , crystallography , stereochemistry , biochemistry , biology , organic chemistry
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 Ca 2+ binds only to E and interior Ca 2+ 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 c ECa 2 , the enzyme with Ca 2+ bound at the exterior site, was measured at different Ca 2+ concentrations with empty sarcoplasmic reticulum vesicles (SRV) and with SRV loaded with 40 mM Ca 2+ by reaction with 0.5 mM [γ‐ 32 P]ATP plus 20 mM EGTA for 13 ms (100 mM KCl, 5 mM MgSO 4 , 40 mM Mops/KOH, pH 7.0, 25°C). The sigmoidal dependence on free exterior calcium concentration of the concentration of c ECa 2 , measured as [ 32 P]phosphoenzyme, is identical with empty and loaded SRV, within experimental error. The value of K 0.5 is 2.8 μM, and the Hill coefficient is 2. This result shows that there is no competition between binding of Ca 2+ 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 Ca 2+ is determined by the state of phosphorylation of the CaATPase.