ATP binding equilibria of the Na(+),K(+)-ATPase

Area of Science:

• Biochemistry
• Enzyme kinetics
• Molecular biology

Background:

• The Na(+),K(+)-ATPase enzyme plays a crucial role in cellular transport.
• Reported dissociation constants (Kd) for ATP binding to the E1 conformation of Na(+),K(+)-ATPase vary significantly between experimental methods.
• This discrepancy in Kd values challenges our understanding of enzyme-ligand interactions.

Purpose of the Study:

• To reconcile the differing Kd values for ATP binding to the Na(+),K(+)-ATPase E1 conformation.
• To investigate the underlying molecular mechanisms causing the observed experimental discrepancies.

Main Methods:

• Computational simulations of enzyme binding models.
• Thermodynamic data analysis using isothermal titration calorimetry.
• Presteady-state kinetic studies and equilibrium binding measurements.

Main Results:

• Excess Mg(2+) ions in kinetic studies can compete with ATP, affecting Kd.
• Inaccurate assumptions about ATP binding kinetics contribute to Kd variations.
• A diprotomer model with two distinct ATP binding affinities explains both equilibrium and kinetic Kd values.

Conclusions:

• The Na(+),K(+)-ATPase functions as an (alphabeta)(2) diprotomer with two ATP binding sites.
• Protein-protein interactions between alpha-subunits create different ATP affinities.
• This structural model resolves the discrepancy between equilibrium and presteady-state kinetic Kd measurements.