ATP Inactivates Hydrolysis of the K+-Sensitive Phosphoenzyme of Kidney Na+, K+-Transport ATPase and Activates that of Muscle Sarcoplasmic Reticulum Ca2+-Transport ATPase1

Abstract

In order to characterize low affinity ATP-binding sites of renal (Na+,K+) ATPase and sarcoplasmic reticulum (Ca2+)ATPase, the effects of ATP on the splitting of the K+-sensitive phosphoenzymes were compared. ATP inactivated the dephosphorylation in the case of (Na+,K+)ATPase at relatively high concentrations, while activating it in the case of (Ca2+)ATPase. When various nucleotides were tested in place of ATP, inactivators of (Na+,K+)ATPase were found to be activators in (Ca2+)ATPase, with a few exceptions. In the absence of Mg2+, the half-maximum concentration of ATP for the inhibition or for the activation was about 0.35 mM or 0.25 mM, respectively. These values are comparable to the previously reported Km or the dissociation constant of the low affinity ATP site estimated from the steady-state kinetics of the stimulation of ATP hydrolysis or from binding measurements. By increasing the concentration of Mg2+, but not Na+, the effect of ATP on the phosphoenzyme of (Na+,K+)ATPase was reduced. On the other hand, Mg2+ did not modify the effect of ATP on the phosphoenzyme of (Ca2+)ATPase. During (Na+,K+)ATPase turnover, the low affinity ATP site appeared to be exposed in the phosphorylated form of the enzyme, but the magnesium-complexed ATP interacted poorly with the reactive K+-sensitive phosphoenzyme, which has a tightly bound magnesium, probably because of interaction between the divalent cations. In the presence of physiological levels of Mg2+ and K+, ATP appeared to bind to the (Na+,K+)ATPase only after the dephosphorylation, while it binds to the (Ca2+)-ATPase before the dephosphorylation to activate the turnover.

Application of the theory of enzyme subunit interactions to ATP-hydrolyzing enzymes. The case of Na,K-ATPase

“…K+ did not affect this effect of ATP. Similarly, Fukushima et al (16) observed the same effect of ATP on phosphoenzyme formed by enzyme + ATP. Furthermore, Norby et al (17), in an analysis of transient kinetic experiments on the dephosphorylation in the presence of K+ of phosphoenzyme formed under Na-ATPase conditions, found that K+ decreased the rate of disappearance of EIP (the first in a sequence of three kinetically isomeric phosphoenzyme forms found in Na-ATPase).…”

“…During the last ten years a number of techniques have been used to investigate whether Na,K-ATPase is, or is not, a functional dimer.' These include the demonstration of apparent "half-of-the-sites" reactivity in phosphorylation experiments (26), when Ca is substituted for Mg, and in cross-linking experiments (27), as well as ligand-induced cooperactivity under equilibrium conditions (12,13,15,16). This evidence has been reviewed by Glynn (6) and by Askari and Huang (28).…”

Interaction of ATP with the Phosphoenzyme of the Na+,K+-ATPase

“…The mechanism by which K þ ions abolish the inhibitory effect of ATP on dephosphorylation cannot be concluded from these studies. However, a feasible hypothesis has been put forward by Fukushima et al (11), who proposed that K þ binding to the phosphoenzyme causes the dissociation of ATP so that it can no longer inhibit dephosphorylation. A critical analysis of this hypothesis would require binding studies to be performed on the phosphoenzyme under conditions where turnover is not possible.…”

“…This must involve ATP binding to the phosphorylated form of the enzyme (7)(8)(9)(10). It has also been found that the dephosphorylation reaction of the Ca 2þ -ATPase is accelerated by ATP, which also necessitates ATP binding to the phosphorylated enzyme (10,11). Thus, these results indicate that phosphorylation and ATP binding without phosphorylation are not mutually exclusive reactions for P-type ATPases; i.e., the enzymes can exist in a state in which they are simultaneously phosphorylated and have bound ATP.…”

Phosphate inhibition of the human red cell sodium pump: simultaneous binding of adenosine triphosphate and phosphate.

“…1) Eisner & Richards, 1983). The alternative pathway may also provide a functional role for enzyme species which bind both phosphate and ATP whose presence has been inferred from binding studies using purified enzyme (Askari & Huang, 1984;Fukushima, Yamada & Nakao, 1984), although the exact relation is unclear since ATP binding to the phosphorylated enzyme retards dephosphorylation without appreciably changing the apparent dissociation constant for phosphate under the conditions of the binding studies. It is worth pointing out that we have proposed (Sachs, 1987) the existence of alternative pathways after E2P2K based on the characteristics of vanadate inhibition of the pump.…”