Reaction sequences for (Na+ + K+)-dependent ATPase hydrolytic activities: new quantitative kinetic models.
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 Pi release. However, contrary to requirements of usual formulations, Pi 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 Pi release, but also requiring a minor alternative pathway with ATP adding after K+ binds but before Pi release. The fit to the data also reveals that Pi binds nearly as rapidly to E2 X K X ATP as to E2 X K, whereas ATP binds quite slowly to E2 X P X K: the site resembles a cul-de-sac with distal ATP and proximal Pi sites. For the K+-nitrophenyl phosphatase reaction also catalyzed by this enzyme, the apparent affinities for both substrate and Pi (as inhibitor) decrease with higher KCl concentrations, and both Pi 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 Pi or TNP-ATP. The greater sensitivity of the phosphatase reaction to both Pi and arsenate is attributable to the weaker binding by the occluded-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 E1/E2 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 E2 conformation as the active site for the phosphatase reaction.
D000818:Animals; D001149:Arsenates / Q000494:pharmacology; D004285:Dogs; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D010710:Phosphates / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011189:Potassium Chloride; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D001149:Arsenates; D010710:Phosphates; D011189:Potassium Chloride; D010744:Phosphoric Monoester Hydrolases; D000254:Sodium-Potassium-Exchanging ATPase