Cooperative Setting for Long-Range Linkage of Ca2+ Bindingand ATP Synthesis in the Ca2+ ATPase

AbstractHigh-affinity and cooperative binding of two Ca2+ per ATPase (SERCA) occurs within the membrane-bound region of the enzyme. Direct measurements of binding at various Ca2+ concentrations demonstrate that site-directed mutations within this region interfere selectively with Ca2+ occupancy of either one or both binding sites and with the cooperative character of the binding isotherms. A transition associated with high affinity and cooperative binding of the second Ca2+ and the engagement of N796 and E309 are both required to form a phosphoenzyme intermediate with ATP in the forward direction of the cycle and also to form ATP from phosphoenzyme intermediate and ADP in the reverse direction of the cycle. This transition, defined by equilibrium and kinetic characterization of the partial reactions of the enzyme cycle, extends from transmembrane helices to the catalytic site through a long-range linkage and is the mechanistic device for interconversion of binding and phosphorylation potentials