| Abstract: | A detailed steady-state kinetic investigation of the hydrolysis of ATP catalyzed by (Na+ + K+)-ATPase is reported. The activity was studied in the presence of (i) Na+ (130 mM), K+ (20 mM) and micromolar ATP concentrations and Na+ (150 mM) the ('Na+-enzyme'). The data obtained lead to the following results: 1. The action of each enzyme may be described by a simple kinetic mechanism with one (Na+-enzyme) or two ((Na+ + K+)-enzyme) dead-end Mg complexes. 2. For both enzymes, both MgATP and free ATP are substrates, with Mg2+, in the latter case, as the second substrate. 3. For each enzyme, the complete set of kinetic constants (seven for the Na+-enzyme, eight for the (Na+ + K+)-enzyme) are determined from the data. 4. For each enzyme it is shown that, in the alternate substrate mechanism obtained, the ratio of net steady-state flux along the 'MgATP pathway' to that of the 'ATP-Mg pathway' increases linearly with the concentration of free Mg2+. The parameters of this function are determined from the data. As a result of this, at high (greater than 3 mM) free Mg2+ concentrations the alternate substrate mechanism degenerates into a 'limiting' kinetic mechanism, with MgATP as the (essentially) sole substrate, and Mg2+ as an uncompetitive (Na+-enzyme) or non-competitive ((Na+ + K+)-enzyme) inhibitor. |
