Substrate-metal interactions at the active site of kidney (Na+ + K+)-ATPase characterized by Mn(II) electron paramagnetic resonance

Abstract Electron paramagnetic resonance spectra at 35 GHz of Mn 2+ ion bound to highly purified membrane-bound (Na + + K + )-ATPase from sheep kidney medulla are much narrower than the corresponding spectra at 9 GHz. As a result, the sensitivity of the enzyme-Mn 2+ spectrum to added substrates is much greater at the higher frequency. ATP and AMP-PNP, which caused very little broadening at low frequency, effect dramatic decreases in intensity of the Mn 2+ EPR signal at 35 GHz. On the other hand, virtually no changes are observed upon addition of ADP and AMP, suggesting that the γ-phosphate of ATP plays a key role in the interaction between Mn 2+ and ATP on the enzyme. The data indicate that ATP and AMP-PNP, binding at low affinity substrate sites, induce a severe distortion of the Mn 2+ coordination geometry. The data also support the suggestion that the enzyme-bound Mn 2+ does not enter into a typical M 2+ -ATP complex in this system.