ATP-ADP exchange reaction catalyzed by Na+,K+-ATPase: dephosphorylation by ADP of the E1P enzyme form.
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.
Campos
M
M
División de Biofísica, Instituto de Investigación Médica "Mercedes y Martín Ferreyra", Casilla de Correo 389, 5000 Córdoba, Argentina.
Beaugé
L
L
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002918:Chymotrypsin / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects / Q000378:metabolism*; D013552:Swine
D002413:Cations, Divalent; D010446:Peptide Fragments; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002918:Chymotrypsin; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium