Phosphorylation of the Sodium−Potassium Adenosinetriphosphatase Proceeds through a Rate-Limiting Conformational Change Followed by Rapid Phosphoryl Transfer

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Abstract
The sodium−potassium adenosinetriphosphatase of sheep kidney, preincubated with sodium and magnesium (E·Na3), reacts with 0.01−2.00 mM ATP to form covalent phosphoenzyme (E-P). The first order rate constant for phosphorylation increases hyperbolically with ATP concentration with a maximum value of (4.6 ± 0.9) × 102 s-1 and K0.5 = 75 ± 25 μM (pH 7.4, 25 °C, 120 mM NaCl, and 3 mM MgCl2). If the phosphoryl-transfer step were rate-limiting, the approach to equilibrium to give 50% E-P in the presence of ADP would follow kobsd = kf + kr = 9.2 × 102 s-1. However, the formation of phosphoenzyme from E·Na3 with 1.0 mM ATP plus 2.0 mM ADP proceeds to 50% completion with kobsd = (4.2 ± 0.8) × 102 s-1. This result shows that phosphoryl transfer from bound ATP to the enzyme is not the rate-limiting step for phosphoenzyme formation from E·Na3. The result is consistent with a rate-limiting conformational change of the E·Na3·ATP intermediate that is followed by rapid phosphoryl transfer, with kcat ≥ 3000 s-1.

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