Solvent effects on substrate and phosphate interactions with the (Na+ + K+)-ATPase.
(Na+ + K+)-ATPase activity of a dog kidney enzyme preparation was markedly inhibited by 10-30% (v/v) dimethyl sulfoxide (Me2SO) and ethylene glycol (Et(OH)2); moreover, Me2SO produced a pattern of uncompetitive inhibition toward ATP. However, K+-nitrophenylphosphatase activity was stimulated by 10-20% Me2SO and Et(OH)2 but was inhibited by 30-50%. Me2SO decreased the Km for this substrate but had little effect on the Vmax below 30% (at which concentration Vmax was then reduced). Me2SO also reduced the Ki for Pi and acetyl phosphate as competitors toward nitrophenyl phosphate but increased the Ki for ATP, CTP and 2-O-methylfluorescein phosphate as competitors. Me2SO inhibited K+-acetylphosphatase activity, although it also reduced the Km for that substrate. Finally, Me2SO increased the rate of enzyme inactivation by fluoride and beryllium. These observations are interpreted in terms of the E1P to E2P transition of the reaction sequence being associated with an increased hydrophobicity of the active site, and of Me2SO mimicking such effects by decreasing water activity: (i) primarily to stabilize the covalent E2P 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 E2P intermediate; but not inhibiting (at lower Me2SO concentrations) the K+-nitrophenylphosphatase reaction that does not pass through such an intermediate; and (ii) secondarily to favor partitioning of Pi and non-nucleotide phosphates into the hydrophobic active site, thereby decreasing the Km for nitrophenyl phosphate and acetyl phosphate, the Ki for Pi 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 E2.P intermediate of that reaction. In addition, Me2SO may decrease binding at the adenine pocket of the low-affinity substrate site, represented as an increased Ki for ATP, CTP and 3-O-methylfluorescein phosphate.
Robinson
J D
JD
Department of Pharmacology, SUNY Health Science Center, Syracuse 13210.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001608:Beryllium / Q000494:pharmacology; D001665:Binding Sites; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004285:Dogs; D019855:Ethylene Glycol; D005026:Ethylene Glycols / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D012997:Solvents; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology; D014867:Water / Q000378:metabolism
D005026:Ethylene Glycols; D010710:Phosphates; D012997:Solvents; D015636:Magnesium Chloride; D014867:Water; D000255:Adenosine Triphosphate; D014508:Urea; D000254:Sodium-Potassium-Exchanging ATPase; D019855:Ethylene Glycol; D008274:Magnesium; D001608:Beryllium; D005459:Fluorides; D004121:Dimethyl Sulfoxide