The effect of di]methylsulfoxide on the substrate site of Na~+/K~+-ATPase studied through phosphorylation by inorganic phosphate and ouabain binding

摘要

To obtain further information on the role of H2O at the substrate site of Na+/K+-ATPase, we have studied the enzymes reaction with Pi and ouabain in 40% (v/v) Me2SO (dimethylsulfoxide). When the enzyme (E) was incubated with ouabain (O) for 5 min in a 40% (v/v) Me2SO-medium with 5 mM MgC12 and 0.5 mM KCl (but no phosphate), ouabain was bound (as EO). Subsequent incubation with Pi showed that E, but not EO, was rapidly phosphorylated (to EP). Long-time phosphorylation revealed that EO is also phosphorylated by Pi albeit very slowly ( about 60 min) and that binding of ouabain to EP also is very slow. The EOP complex is stable, i.e., the for the loss of Pi is 60 min in contrast to about 1 min in water. These results in 40% Me2SO are distinctly different from what would be obtained in a watery milieu: ouabain would bind slowly and inefficiently in the absence of Pi, and ouabain would catalyse phosphorylation from Pi rather than retard it. Equilibrium binding of [3H]ouabain to E and EP in water or 40% Me,SO confirmed these observations: Kdiss in water is 11 μM and 12 nM for EO and EOP, respectively, whereas in Me2SO they are 112 nM and 48 nM. It is suggested that the primary effect of the lowered water activity in 40% Me2SO is a rearrangement of the substrate site so that it also in the absence of Pi attains a transition state configuration corresponding to the phosphorylated conformation. This would be sensed by the ouabain binding site and lead to high affinity ouabain binding in the absence of Pi.