Solvent effects on substrate and phosphate interactions with the (Na + + K +)-ATPase

Abstract

(Na + + K +)-ATPase activity of a dog kidney enzyme preparation was markedly inhibited by 10–30% (v/v) dimethyl sulfoxide (Me 2SO) and ethylene glycol (Et(OH) 2); moreover, Me 2SO produced a pattern of uncompetitive inhibition toward ATP. However, K +-nitrophenylphosphatase activity was stimulated by 10–20% Me 2SO and Et(OH) 2 but was inhibited by 30–50%. Me 2SO decreased the K m for this substrate but had little effect on the V max below 30% (at which concentration V max was then reduced). Me 2SO also reduced the K i for P i and acetyl phosphate as competitors toward nitrophenyl phosphate but increased the K i for ATP, CTP and 3- O-methylflourescein phosphate as competitors. Me 2SO inhibited K +-acetylphosphatase activity, although it also reduced the K m for that substrate. Finally, Me 2SO increased the rate of enzyme inactivation by fluoride and beryllium. These observations are interpreted in terms of the E 1P to E 2P transition of the reaction sequence being associated with an increased hydrophobicity of the active site, and of Me 2SO mimicking such effects by decreasing water activity: (i) primarily to stabilize the covalent E 2P intermediate, through differential solvation of reactants and products, and thereby inhibiting the (Na + + K +)-ATPase reaction and acting as a dead-end inhibitor to produce the pattern of uncompetitive inhibition; inhibiting the K +-acetylphosphatase reaction that also passes through an E 2P intermediate; but not inhibiting (at lower Me 2SO concentrations) the K +-nitrophenylphosphatase reaction that does not pass through such an intermediate; and (ii) secondarily to favor partitioning of P i and non-nucleotide phosphates into the hydrophobic active site, thereby decreasing the K m for nitrophenyl phosphate and acetyl phosphate, the K i for P i and acetyl phosphate in the K +-nitrophenylphosphatase reaction, accelerating inactivation by fluoride and beryllium acting as phosphate analogs, and, at higher concentrations, inhibiting the K +-nitrophenylphosphatase reaction by stabilizing the non-covalent E 2·P intermediate of that reaction. In addition, Me 2SO may decrease binding at the adenine pocket of the low-affinity substrate site, represented as an increased K i for ATP, CTP and 3- O-methylfluorescein phosphate.