KINETICS OF K-STIMULATED DEPHOSPHORYLATION AND SIMULTANEOUS K+ OCCLUSION BY NA,K-ATPASE, STUDIED WITH THE K+ CONGENER TL+ - THE POSSIBILITYOF DIFFERENCES BETWEEN THE 1ST TURNOVER AND STEADY-STATE()

Using the K+ congener Tl+ and rapid mixing combined with special quenc h techniques, we have investigated (at 20-degrees-C) what is usually a ssumed to be the enzymatic correlate of active transport of K+ by Na,K -ATPase: the Tl+-catalyzed dephosphorylation of the K+-sensitive phosp hointermediate(s), EP, and the resulting occlusion of Tl+ in the enzym e protein. We measured [EP] and [occluded TI] as a function of time in phosphorylation, as well as dephosphorylation experiments with the fo llowing 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 ph osphorylation rate. Tl+-catalyzed dephosphorylation and Tl+ occlusion appeared to be simultaneous, and the stoichiometry was always 2 Tl+ oc cluded/EP dephosphorylated. Second, we tried computer simulations of t he transient kinetic experiments, using an Albers-Post-type reaction s cheme. This produced satisfactory curve-fits only in the case of 150 m M Na+, and although we could arrange that calculated [EP]steady-state was equal to that measured, the calculated steady-state Na,Tl-ATPase h ydrolysis rates were always two to four times the rates measured direc tly. Third, we propose, as one (possibly of several) solution to these discrepancies between model and data, an expanded kinetic model consi sting of an initiation reaction sequence followed by a propagation (or steady-state) reaction cycle. In this alternative model the first tur nover 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.