Inactivation of Na,K-ATPase following Co(NH₃)₄ATP binding at a low affinity site in the protomeric unit

Douglas Ward, JD Cavieres

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The Na+-dependent or E1 stages of the Na,K-ATPase reaction require a few micromolar ATP, but submillimolar concentrations are needed to accelerate the K+-dependent or E2 half of the cycle. Here we use Co(NH3)4ATP as a tool to study ATP sites in Na,K-ATPase. The analogue inactivates the K+ phosphatase activity (an E2 partial reaction) and the Na,K-ATPase activity in parallel, whereas ATP-[3H]ADP exchange (an E1 reaction) is affected less or not at all. Although the inactivation occurs as a consequence of low affinity Co(NH3)4ATP binding (KD ≈ 0.4–0.6 mm), we can also measure high affinity equilibrium binding of Co(NH3)4[3H]ATP (KD = 0.1 μm) to the native enzyme. Crucially, we find that covalent enzyme modification with fluorescein isothiocyanate (which blocks E1 reactions) causes little or no effect on the affinity of the binding step preceding Co(NH3)4ATP inactivation and only a 20% decrease in maximal inactivation rate. This suggests that fluorescein isothiocyanate and Co(NH3)4ATP bind within different enzyme pockets. The Co(NH3)4ATP enzyme was solubilized with C12E8 to a homogeneous population of αβ protomers, as verified by analytical ultracentrifugation; the solubilization did not increase the Na,K-ATPase activity of the Co(NH3)4ATP enzyme with respect to parallel controls. This was contrary to the expectation for a hypothetical (αβ)2 membrane dimer with a single ATP site per protomer, with or without fast dimer/protomer equilibrium in detergent solution. Besides, the solubilized αβ protomer could be directly inactivated by Co(NH3)4ATP, to less than 10% of the control Na,K-ATPase activity. This suggests that the inactivation must follow Co(NH3)4ATP binding at a low affinity site in every protomeric unit, thus still allowing ATP and ADP access to phosphorylation and high affinity ATP sites.
Original languageEnglish
Pages (from-to)14688-14697
Number of pages10
JournalJournal of Biological Chemistry
Issue number17
Publication statusPublished - 18 Apr 2003


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