Evidence for a Mg2+‐Induced Conformational Change at the ATP‐Binding Site of (Na++ K+)‐ATPase Demonstrated with a Photoreactive ATP‐Analogue

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Abstract
The 3''-ribosyl ester of ATP with 2-nitro-4-azidophenyl propionic acid was prepared and its ability to act as a photoaffinity label of (Na+ + K+)-ATPase [from pig kidney] was tested. In the dark 3''-O-[3-(2-nitro-4-azidophenyl)propionyl]ATP (N3-ATP) is a substrate of (Na+ + K+)-ATPase and a competitive inhibitor of ATP hydrolysis. Upon irradiation by UV light, N3-ATP photolabeled the high-affinity ATP-binding site and was covalently attached to the .alpha.-subunit and an approximately 12,000-Mr [relative MW] component. Photolabeling of the .alpha.-subunit by N3-ATP irreversibly inactivated (Na+ + K+)-ATPase. Photoinactivation was strictly Mg2+-dependent. Na+ enhanced the inactivation. ATP or ADP and K+ protected the enzyme against inactivation. Mg2+, in concentrations required for photoinactivation, protected (Na+ + K+)-ATPase against inactivation by tryptic digestion under controlled conditions. A conformational change of the ATP-binding site of (Na+ + K+)-ATPase may occur upon binding of Mg2+ to a low-affinity site.
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