The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na + + K +)-ATPase from ox brain I. Substrate identity

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 millimolar ATP concentrations (the ‘( Na + + K +)- enzyme’ ), as well as (ii) with micromolar ATP concentrations and Na + (150 mM) (the ‘Na +-enzyme’). The data obtained lead to the following results: 1. 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. 2. For both enzymes, both MgATP and free ATP are substrates, with Mg 2+, in the latter case, as the second substrate. 3. 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. 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 Mg 2+. The parameters of this function are determined from the data. As a result of this, at high (greater than 3 mM) free Mg 2+ concentrations the alternate substrate mechanism degenerates into a ‘limiting’ kinetic mechanism, with MgATP as the (essentially) sole substrate, and Mg 2+ as an uncompetitive (Na +-enzyme) or non-competitive ((Na + + K +)-enzyme) inhibitor.