THE EFFECT OF DIMETHYLSULFOXIDE ON THE SUBSTRATE SITE OF NA+ K+-ATPASE STUDIED THROUGH PHOSPHORYLATION BY INORGANIC-PHOSPHATE AND OUABAIN BINDING/

To obtain further information on the role of H2O at the substrate site of Na+/K+-ATPase, we have studied the enzymes reaction with P-i and o uabain in 40% (v/v) Me(2)SO (dimethylsulfoxide). When the enzyme (E) w as incubated with ouabain (O) for 5 min in a 40% (v/v) Me(2)SO-medium with 5 mM MgCl2 and 0.5 mM KCl (but no phosphate), ouabain was bound ( as EO). Subsequent incubation with P-i showed that E, but not EO, was rapidly phosphorylated (to EP). Long-time phosphorylation revealed tha t EO is also phosphorylated by P-i albeit very slowly (t(1/2) about 60 min) and that binding of ouabain to EP also is very slow. The EOP com plex is stable, i.e., the t(1/2) for the loss of P-i is much greater t han 60 min in contrast to about 1 min in water. These results in 40% M e(2)SO are distinctly different from what would be obtained in a water y milieu: ouabain would bind slowly and inefficiently in the absence o f P-i, and ouabain would catalyse phosphorylation from P-i rather than retard it. Equilibrium binding of [H-3]ouabain to E and EP in water o r 40% Me(2)SO confirmed these observations: K-diss in water is 11 mu M and 12 nM for EO and EOP, respectively, whereas in Me(2)SO they are 1 12 nM and 48 nM. It is suggested that the primary effect of the lowere d water activity in 40% Me(2)SO is a rearrangement of the substrate si te so that it also in the absence of P-i attains a transition state co nfiguration corresponding to the phosphorylated conformation. This wou ld be sensed by the ouabain binding site and lead to high affinity oua bain binding in the absence of P-i.