Kinetics of Na+-ATPase: influence of Na+ and K+ on substrate binding and hydrolysis.
An analysis of the influence of Na+ and K+ on the kinetics of Na+-ATPase in broken membrane preparations from bovine brain is presented with particular emphasis on the effect of the cations on the binding and splitting of the substrate MgATP and on the derivation of a detailed kinetic model for that interaction. It was found that the enzyme in the absence of Na+ and K+, but in the presence of 7 mM free Mg2+, at pH 7.4 (37 degrees C) exhibits an ouabain-sensitive ATPase activity. The simplest model quantitatively compatible with all the data involves two different, interconvertible (conformational) forms of the enzyme, E1 and E'1, with the following properties: The E1 form does not bind K+ but has three independent and equivalent high-affinity sites (Kd = 5.6 mM) for Na+. It binds and hydrolyzes substrate only when two or three sodium ions are bound to it. The E'1 form binds and hydrolyzes the substrate only in the absence of monovalent cations. It is competitively inhibited by K+ (Kd = 0.23 mM), and this inhibition is further enhanced by binding of Na+ to the K+-bound form at two equivalent, independent sites (Kd = 12 mM). It is suggested that the E'1 form is the Mg2+-induced conformational state of the enzyme observed by others, which differs from the usually encountered E1 and E2 forms. The model allows the calculation of ATP-binding and ADP-releasing rate constants for the E1-form for later comparison with corresponding rate constants for the (na+ + K+)-ATPase (following paper).
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001921:Brain / Q000201:enzymology; D027682:Cation Transport Proteins; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008433:Mathematics; D008956:Models, Chemical; D011188:Potassium / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology*; D013379:Substrate Specificity
D027682:Cation Transport Proteins; D012964:Sodium; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase; D011188:Potassium