Effects of magnesium and ATP on pre-steady-state phosphorylation kinetics of the Na+,K(+)-ATPase.
The aim of the present work was to elucidate the role played by ATP and Mg2+ ions in the early steps of the Na+,K(+)-ATPase cycle. The approach was to follow pre-steady-state phosphorylation kinetics in Na(+)-containing K(+)-free solutions under variable ATP and MgCl2 concentrations. The experiments were performed with a rapid mixing apparatus at 20 +/- 2 degrees C. The concentrations of free and complexes species of Mg2+ and ATP were calculated on the basis of a dissociation constant of 0.091 +/- 0.004 mM, estimated with Arsenazo III under identical conditions. A simplified scheme were ATP binds to the ENa enzyme, which is phosphorylated to MgEPNa and consequently dephosphorylated returning to the ENa form, was used. In the absence of ADP and phosphate four rate constants are relevant: k1 and k-1, the on and off rate constants for ATP binding; k2, the transphosphorylation rate constant and k3, the constant that governs the dephosphorylation rate. The values obtained were: k1 = 0.025 +/- 0.003 microM-1 ms-1 for both free ATP and ATPMg; k-1 = 0.038 +/- 0.004 ms-1 for free ATP and 0.009 +/- 0.002 ms-1 for ATPMg; k2 = 0.199 +/- 0.005 ms-1; k3 = 0.0019 +/- 0.0002 ms-1. The model that seems best to explain the data is one where (i) the role of true substrate can be played equally well by free ATP or ATPMg, and (ii) free Mg2+, an essential activator, acts by binding to a specific Mg2+ site on the enzyme molecule.
Campos
M
M
División de Biofísica, Instituto de Investigación Médica M. y M. Ferreyra, Córdoba, Argentina.
Beaugé
L
L
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D002413:Cations, Divalent; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium