Kinetics of K(+)-stimulated dephosphorylation and simultaneous K+ occlusion by Na,K-ATPase, studied with the K+ congener Tl+. The possibility of differences between the first turnover and steady state.

Using the K+ congener Tl+ and rapid mixing combined with special quench techniques, we have investigated (at 20 degrees C) what is usually assumed to be the enzymatic correlate of active transport of K+ by Na,K-ATPase: the Tl(+)-catalyzed dephosphorylation of the K(+)-sensitive phosphointermediate(s), EP, and the resulting occlusion of Tl+ in the enzyme protein. We measured [EP] and [occluded Tl] as a function of time in phosphorylation, as well as dephosphorylation experiments with the following results. First, we found that with 150 mM Na+ and 600 mM Na(+)--NO3- was the anion--[Tl+] = 0.1-1 mM was without influence on the phosphorylation rate. Tl(+)-catalyzed dephosphorylation and Tl+ occlusion appeared to be simultaneous, and the stoichiometry was always 2 Tl+ occluded/EP dephosphorylated. Second, we tried computer simulations of the transient kinetic experiments, using an Albers-Post-type reaction scheme. This produced satisfactory curve-fits only in the case of 150 mM Na+, and although we could arrange that calculated [EP]steady-state was equal to that measured, the calculated steady-state Na,Tl-ATPase hydrolysis rates were always two to four times the rates measured directly. Third, we propose, as one (possibly of several) solution to these discrepancies between model and data, an expanded kinetic model consisting of an initiation reaction sequence followed by a propagation (or steady-state) reaction cycle. In this alternative model the first turnover of the enzyme is kinetically different from subsequent reaction cycles, and this allowed us to obtain both satisfactory curve-fits and accordance between calculated and measured values of hydrolysis-rate and [EP]steady-state.