Ca2+ binding to sarcoplasmic reticulum ATPase revisited. II. Equilibrium and kinetic evidence for a two-route mechanism.

The experiments reported in the present paper were designed to check the model proposed for Ca2+ binding in the preceding paper (Forge, V., Mintz, E., and Guillain, F. (1993) J. Biol. Chem. 268, 10953-10960). The pH dependence of the Mg(2+)-induced variation of the intrinsic fluorescence, as well as that of the phosphorylation by Pi, confirmed that there are several species of Ca(2+)-deprived ATPase. Kinetics of Ca2+ binding as a function of pH suggested that the deprotonated form of the ATPase binds Ca2+ rapidly (k > 50 s-1), whereas the protonated forms bind Ca2+ slowly (1.3-2.7 s-1). At variance with other models which are linear, slow and rapid Ca2+ binding take two different routes, and intermediate pH values and Mg2+, which favors the deprotonated forms, result in biphasic kinetics. Mg2+ binds to all Ca(2+)-deprived species and to species having one bound Ca2+ but does not bind to ECa2. This is the reason why Mg2+ inhibits Ca2+ binding, and this inhibition is removed in the presence of adenosine-5'-O-(3-thiotriphosphate) which drives Mg2+ into the catalytic site.