Low affinity acceleration of the phosphorylation reaction of the Na,K-ATPase by ATP.

The maximum rate of phosphorylation (rm) of a highly purified Na,K-ATPase from red outer medulla of pig kidney was measured at 25 degrees C as a function of ATP concentration in media with Mg2+, Na+, and no K+. When rm was plotted as a function of the concentration of ATP a biphasic response was observed with a hyperbolic component of high affinity (Km = 15.7 +/- 2.6 microM) and low velocity ((rm)max = 460 +/- 40 nmol of Pi/(mg of protein.s)) plus a parabolic component which showed no saturation up to 1000 microM ATP, concentration at which rm was 1768.1 +/- 429.6 nmol Pi/(mg protein.s) (mean +/- S.E.; n = 3). This low affinity effect of ATP on the rate of phosphorylation disappeared when the Na,K-ATPase underwent turnover in medium without K+ suggesting that, like superphosphorylation (Peluffo, R. D., Garrahan, P. J., and Rega, A. F. (1992) J. Biol. Chem. 267, 6596-6601), it required the enzyme to be at rest. This property of the Na,K-ATPase was not predicted by the Albers-Post reaction scheme. The observed behavior of the enzyme could be simulated by a scheme that involves a resting enzyme (Er) functionally different from E1 or E2, which is able to bind three molecules of ATP, one with high and two with low affinity, and that after phosphorylation is converted into the phosphointermediates that are generally considered to participate in the reaction cycle described by Albers and Post.