Phosphorus-31 nuclear magnetic resonance of phosphoenzymes of sodium- and potassium-activated and of calcium-activated adenosinetriphosphatase.
Prior studies identified phosphoenzyme intermediates in the turnover of sodium- and potassium-activated adenosinetriphosphatase [(Na,K)ATPase] from several sources and of the calcium-activated adenosinetriphosphatase [(Ca)-ATPase] of skeletal muscle sarcoplasmic reticulum. In both cases, the transphosphorylation is to a beta-aspartyl carboxyl group at the active site. We now report observation of a K+-sensitive phosphorylated intermediate of purified (Na,-K)ATPase from the salt gland of the duck using high-field 31P nuclear magnetic resonance. Addition of ATP to a suspension of this enzyme in the presence of Mg2+ and Na+ produced a resonance at about +17 ppm relative to 85% phosphoric acid. Addition of inorganic phosphate and Mg2+ to (Na,K)ATPase also produced a resonance at about +17 ppm which was enhanced in the presence of a saturating concentration of the inhibitor, ouabain; again, addition of K+ made this resonance disappear. These findings are consistent with earlier kinetic characterization of an acid-stable (Na,K)ATPase phosphoenzyme intermediate by 32P-labeled phosphate incorporation into a denatured precipitate of the enzyme. We attribute the +17-ppm resonance to formation of an acyl phosphate at an aspartyl residue of the catalytic site of (Na,K)ATPase. This is supported by our finding of a similar resonance at +17 ppm after phosphorylation of another membrane-bound cation transport enzyme, sarcoplasmic reticulum (Ca)ATPase, as well as by a similar resonance at about +17 ppm after phosphorylation of the model dipeptide L-seryl-L-aspartate.
D000818:Animals; D000252:Calcium-Transporting ATPases; D004372:Ducks; D009682:Magnetic Resonance Spectroscopy; D009132:Muscles / Q000201:enzymology; D010750:Phosphoproteins; D010766:Phosphorylation; D011817:Rabbits; D012491:Salt Gland / Q000201:enzymology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D000254:Sodium-Potassium-Exchanging ATPase
D010750:Phosphoproteins; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase