Identification of Potential Regulatory Sites of the Na+,K+-ATPase by Kinetic Analysis

Kinetic models are presented that allow the Na+,K+-ATPase steady-state turnover number to be estimated at given intra- and extracellular concentrations of Na+, K+, and ATP. Based on experimental transient kinetic data, the models utilize either three or four steps of the Albers−Post scheme, that is, E2 → E1, E1 → E2P (or E1 → E1P and E1P → E2P), and E2P → E2, which are the major rate-determining steps of the enzyme cycle. On the time scale of these reactions, the faster binding steps of Na+, K+, and ATP to the enzyme are considered to be in equilibrium. Each model was tested by comparing calculations of the steady-state turnover from rate constants and equilibrium constants for the individual partial reactions with published experimental data of the steady-state activity at varying Na+ and K+ concentrations. To provide reasonable agreement between the calculations and the experimental data, it was found that Na+/K+ competition for cytoplasmic binding sites was an essential feature required in the model. The activity was also very dependent on the degree of K+-induced stimulation of the reverse reaction E1 → E2. Taking into account the physiological substrate concentrations, the models allow the most likely potential sites of short-term Na+,K+-ATPase regulation to be identified. These were found to be (a) the cytoplasmic Na+ and K+ binding sites, via changes in Na+ or K+ concentration or their dissociation constants, (b) ATP phosphorylation (as a substrate), via a change in its rate constant, and (c) the position of the E2 ⇌ E1 equilibrium.