ATP−ADP Exchange Reaction Catalyzed by Na⁺,K⁺-ATPase:  Dephosphorylation by ADP of the E₁P Enzyme Form

Abstract

We studied the effects of Mg2+ and of ADP and other nucleoside diphosphates on the dephosphorylation of the E1P form of the partially purified pig kidney Na+,K+-ATPase at 20-22 degrees C. We report for the first time the rate of the reversal of ATP phosphorylation. The experiments were done on enzyme subjected to controlled chymotrypsin digestion consisting of a homogenous population of a truncated catalytic subunit. Under this condition the whole cycle is E1 <-- (f1.ATP, b1) --> E1ATP <-- (f2, b2) --> E1P.ADP <-- (fd, bd.ADP) --> E1P-(f3) --> E1. The values of f1, b1, f2, and f3 were independently estimated in the absence of ADP; those of fd, bd, and b2 were obtained from the fit of ADP-dependent dephosphorylation data to the differential equation set. When f2 = 0 or b1 is very large, the model predicts that dephosphorylation by ADP gives a single exponential; in all other cases it predicts a biphasic dephosphorylation in a semilogarithmic plot. The fast phase is governed by b2.ADP and the slow one by b1. This was experimentally verified. Also, ADP stimulates E1P breakdown without release of Pi, thus leading to ATP synthesis. The data indicate that the true substrate for ATP synthesis is free ADP, while Mg2+ inhibits mainly by a reduction in the free [ADP]; in addition, E1P has a very low affinity for MgADP. The nucleotide structure is also very important; all ADP analogues tested were much less effective than ADP due to a reduced affinity for the E1P and a poor capacity to reverse phosphorylation.

Effect of ADP on Na+-Na+ Exchange Reaction Kinetics of Na,K-ATPase

“…Our K d was consistent with the range of K s ADP values (1-6 mM) reported by Suzuki and Post (1997). Chymotrypsin-modified enzyme, on the other hand, yielded a K d ¼ 37 mM (Campos and Beaugé, 1997; K s at 2 mM Mg 21 ). Whether this value represents a true K d for ADP in the absence of Na 1 -Na 1 exchange or the proteolytic treatment affected ADP binding sites in E 1 ;P, remains to be determined.…”

“…The value of the rate constant for ADP release, k 1 (404 s ÿ1 ), determined under conditions that strongly favor enzyme phosphorylation, is in fair agreement with that reported by Campos and Beaugé (1997) in chymotrypsin-digested enzyme (1067 s ÿ1 , 22°C). Both of these values agree with suggestions that (Na 3 )E 1 ;PÁADP is a short-lived intermediate in the Na,K-ATPase reaction cycle (Mårdh and Post, 1977;Nørby et al, 1983;Hobbs et al, 1985).…”

“…In this regard, ADP has been shown to act as an acceptor of the phosphate group in the phosphoenzyme to yield ATP in a reaction called transphosphorylation or ATP/ADP exchange (Glynn, 1985). A crucial intermediate for ATP/ADP exchange that has been extensively included in the formulation of models to describe ion transport by the Na,K-ATPase is (Na 3 )E 1 ;PÁADP (De Weer, 1970Karlish et al, 1978;Cornelius and Skou, 1985;Kennedy et al, 1986;Forbush and Klodos, 1991;Pratap et al, 1991;Keillor and Jencks, 1996;Suzuki and Post, 1997;Campos and Beaugé, 1997). Nevertheless, this phosphorylated intermediate containing occluded Na ions and bound ADP appears to exist in very small amounts during steady-state Na,K-ATPase cycling (Nørby et al, 1983) and, thus, its kinetic characterization has remained elusive.…”

“…Nevertheless, this phosphorylated intermediate containing occluded Na ions and bound ADP appears to exist in very small amounts during steady-state Na,K-ATPase cycling (Nørby et al, 1983) and, thus, its kinetic characterization has remained elusive. As an approach to solve this problem, Campos and Beaugé (1997) studied ATP/ADP exchange in partially purified Na,K-ATPase subjected to chymotrypsin digestion to estimate the kinetics of ADP binding to and release from the phosphoenzyme. However, proteolytic treatment prevents phosphoenzyme conformational transitions and Na 1 -Na 1 exchange, so that it remains unclear how these rate constants relate to those of the native enzyme.…”

Breakdown of Na+/K+‐exchanging ATPase phosphoenzymes formed from ATP and from inorganic phosphate during Na+‐ATPase activity.

“…In this step, ATP and Na + bind to high‐affinity sites (less than 1 µ m and 1 m m , respectively). ADP is very rapidly released after phosphoenzyme formation [3] but remains able to rebind and push the reaction back into ATP synthesis. Next, MgE 1 P(Na 3 ) is spontaneously converted into MgE 2 PNa 3 ; this form does not bind ADP, and it is from it that three Na + ions are expelled into the extracellular environment.…”