Binding and transport of calcium by sarcoplasmic reticulum ATPase

Abstract Sarcoplasmic Reticulum (SR) membrane can be isolated from striated muscle in the form of sealed vesicles containing a high density of ATPase protein that accounts for approximately half the membrane mass. The ATPase polypeptide units are composed of polar segments protruding from the outer surface of the membrane into the aqueous medium, and hydrophobic segments intruding the membrane bilayer. The specific function of the SR ATPase id to take up Ca2+ form the medium outside the vesicles, and release it into the aqueous medium inside the vesicles. Thereby a transmembrane Ca2+ gradient is formed, deriving free energy from ATP hydrolysis. Activation of the enzyme is totally dependent on calcium binding to high affinity sites which are exposed to the medium outside the vesicles. In the absence of ATP, the calcium binding properties of the SR ATPase can be characterized at equilibrium by measuring the distribution of radioactive calcium tracer, or measuring ensuing changes of protein intrinsic fluorescence. Specific calcium binding (producing enzyme activation) involves two sites per enzyme unit, with a ∼106M−1 apparent association constant, at neutral pH. The relationship of Ca2+ binding to Ca2+ concentration is pH dependent, inasmuch as higher affinity for Ca2+ and higher cooperativity are observed as the H+ concentration is reduced. The experimental data can be fitted satisfactorily assuming competition of one Ca2+ with one H+ for each site, and cooperative interaction of binding sites. When ATP is added to Ca2 ATPase, the ATP terminal phosphate is rapidly transferred to an aspartyl residue of the catalytic site, forming an anhydride bond. Following the phosphorylation reaction the affinity of the sites for Ca2+ is reduced of ∼3 orders of magnitude (K≅ 103M−1), and their orientation is changed to permit dissociation of Ca2+ inside the vesicles. The phosphoenzyme then undergoes hydrolytic cleavage, and the enzyme is free to undergo a new catalytic and transport cycle. When the Ca2+ concentration inside the vesicle is increased ∼3 orders of magnitude, Ca2+ accumulation reaches an asymptote which is permitted by the free energy of ATP, and limited by the dissociation constant acquired by the calcium sites when the enzyme is phosphorylated. Therefore, it is clear that a basic feature of the transport mechanism is a special relationship between binding sites and catalytic site, whereby occupancy of binding sites by Ca2+ is required for the utilization of ATP by catalytic site, and phosphorylation of the catalytic site reduces the affinity of the binding sites for Ca2+. Thereby ATP phosphorylation potential is transformed into Ca2+ concentration potential. The ATPase catalytic site can be phosphorylated not only by ATP, but also by orthophosphate. The latter reaction, however, occurs only in the absence of Ca2+, indicating that phosphorylation of the catalytic site does not require free energy input (i.e., ATP) if the binding sites are free of Ca2+. It is of interest that orthovanadate can form a stable complex with the catalytic site even in the presence of Ca2+ and, in analogy to ATP, it reduces the affinity of the binding sites causing dissociation of Ca2+. Contrary to ATP, however, the vanadate ineraction with the catalytic site is rather stable and does not produce Ca2+ fluxes. It is possible that the vanadate trigonal bipyramidal structure is a stable stereo-analogue of a pentacovalent transition state in the phosphoryl transfer reaction. In addition to the anhydride bond with the aspartyl residue, the vanadate enzyme complex is likely to be stabilized by conformational fit in the protein site, and acceptance of electrons by the vanadate d orbitals from neighboring oxygens. The resulting free energy well prevents cycling of free enzyme and Ca2+ fluxes. The vanadate reaction demonstrates that it is possible to affect the binding characteristics of the calcium binding sites by means other than ATP. The uniqueness of ATP however, resides in its kinetic as well as its thermodynamic adequacy.