Pseudosubstrate hydrolysis by the erythrocyte plasma membrane Ca2+-ATPase: Kinetic evidence for a modified E(1) conformation in dimethylsulfoxide

The purified Ca2+-ATPase of pig red cells displays a phosphatase activity towards p-nitrophenylphosphate which is inhibited by Ca2+ in the absence of solvents, and activated by calmodulin. This activity has been attributed to the E(2) conformation of the enzyme. Here we show that the pNPPase activity in the absence of Ca2+ is stimulated 10-25-fold by the presence of the organic solvent dimethylsulfoxide (Me(2)SO). This is an activation that surpasses by severalfold that induced by calmodulin in the absence of the solvent. At 30% Me(2)SO, activation by calmodulin disappears. In the absence of calmodulin and at pH 7.2, the Ca2+ concentration needed for half-maximal inhibition of the pNPPase activity (K-i) increases from 130 mu M in the absence of Me(2)SO to 860 mu M at 30% Me(2)SO. This effect of Me(2)SO is enhanced at pH 8.0: the K-i for Ca2+ increases from 2.7 mu M ill the absence of the solvent to 2.0 mM in its presence. However, the K-0.5 for Ca2+ activation of the ATPase activity decreases from 8.3 to 2.6 mu M following addition of the same Me(2)SO concentration. This indicates that, even in the presence of Me(2)SO, mu M Ca2+ concentrations shift the equilibrium towards E(1) but the decrease in activity that would be expected if pNPP hydrolysis were catalysed exclusively by the E(2) conformation is not observed. The affinity for pNPP as a substrate increases from 2.6 mM in the absence of Me(2)SO to 1.6 mM in the presence of 20% Me(2)SO. These results suggest that Me(2)SO induces multiple effects in the Ca2+-ATPase that: (i) increase the reactivity of E(2) towards substrate; (ii) surpass the activation by calmodulin and, (iii) allow the enzyme to hydrolyze pNPP even when Ca2+ is bound to the high-affinity sites of the enzyme. The change in reactivity is attributed to an increase on substrate catalysis rather than on pNPP binding.