Reaction sequences for (Na + + K +)-dependent ATPase hydrolytic activities: new quantitative kinetic models

Abstract

To delineate better the reaction sequence of the (Na + + K +)-ATPase and illuminate properties of the active site, kinetic data were fitted to specific quantitative models. For the (Na + + K +)-ATPase reaction, double-reciprocal plots of velocity against ATP (in the millimolar range), with a series of fixed KCl concentrations, are nearly parallel, in accord with the ping pong kinetics of ATP binding at the low-affinity sites only after P i release. However, contrary to requirements of usual formulations, P i is not a competitor toward ATP. A new steady-state kinetic model accommodates these data quantitatively, requiring that under usual assay conditions most of the enzyme activity follows a sequence in which ATP adds after P i release, but also requiring a minor alternative pathway with ATP adding after K + binds but before P i release. The fit to the data also reveals that P i binds nearly as rapidly to E 2 · K · ATP as to E 2 · K, whereas ATP binds quite slowly to E 2 · P · K: the site resembles a cul-de-sac with distal ATP and proximal P i sites. For the K +-nitrophenyl phosphatase reaction also catalyzed by this enzyme, the apparent affinities for both substrate and P i (as inhibitor) decrease with higher KCl concentrations, and both P i and TNP-ATP appear to be competitive inhibitors toward substrate with 10 mM KCl but noncompetitive inhibitors with 1 mM KCl. These data are accommodated quantitatively by a steady-state model allowing cyclic hydrolytic activity without obligatory release of K +, and with exclusive binding of substrate vs. either P i or TNP-ATP. The greater sensitivity of the phosphatase reaction to both P i and arsenate is attributable to the weaker binding by the occuluded-K + enzyme form occurring in the (Na + + K +)-ATPase reaction sequence. The steady-state models are consistent with cyclical interconversion of high- and low-affinity substrate sites accompanying E 1/E 2 transitions, with distortion to low-affinity sites altering not only affinity and route of access but also separating the adenine- and phosphate-binding regions, the latter serving in the E 2 conformation as the active site for the phosphatase reaction.