Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase

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Abstract
The (Na+ + K+)-dependent ATPase exhibits substrate sites with both high affinity (K m near 1 µM) and low affinity (K m near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl2, the nonhydrolyzable β-γ imido analog of ATP, AMP-PNP, was used in experiments performed at 0–4°C by a centrifugation technique. By this method theK D for AMP-PNP was 4.2 µM in the absence of MgCl2. Adding 50 µM MgCl2, however, decreased theK D to 2.2 µM; by contrast, higher concentrations of MgCl2 increased theK D until, with 2 mM MgCl2, theK D was 6 µM. The half-maximal effect of MgCl2 on increasing theK D occurred at approximately 1 mM. This biphasic effect of MgCl2 is interpreted as Mg2+ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl2 concentrations would represent Mg2+ acting through the low-affinity substrate sites. NaCl in the absence of MgCl2 increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl2, however, NaCl increased theK D for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl2, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K+-dependent phosphatase reaction also catalyzed by this enzyme,p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.