LOW-AFFINITY ACCELERATION OF THE PHOSPHORYLATION REACTION OF THE NA,K-ATPASE BY ATP

The maximum rate of phosphorylation (r(m)) of a highly purified Na,K-A TPase 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 r(m) was plotted as a function of the concentration of ATP a b iphasic response was observed with a hyperbolic component of high affi nity (K(m) = 15.7 +/-2.6 muM) and low velocity ((r(m))max = 460 +/- 40 nmol of P(i)/(mg of protein . s)) plus a parabolic component which sh owed no saturation up to 1000 muM ATP, concentration at which r(m) was 1768.1 +/- 429.6 nmol P(i)/(mg protein.s) (mean +/- S.E.; n = 3). Thi s low affinity effect of ATP on the rate of phosphorylation disappeare d when the Na,K-ATPase underwent turnover in medium without K+ suggest ing that, like superphosphorylation (Peluffo, R. D., Garrahan, P. J., and Rega, A. F. (1992) J. Biol. Chem. 267, 6596-660 1), it required th e enzyme to be at rest. This property of the Na,K-ATPase was not predi cted by the Albers-Post reaction scheme. The observed behavior of the enzyme could be simulated by a scheme that involves a resting enzyme ( E(r)) functionally different from E1 or E2, which is able to bind thre e 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 describe d by Albers and Post.