Solvent effects on substrate and phosphate interactions with the (Na+ + K+)-ATPase

Area of Science:

• Biochemistry
• Enzyme kinetics
• Membrane transport

Background:

• The (Na+ + K+)-ATPase enzyme is crucial for maintaining cellular ion gradients.
• Understanding its regulation by small molecules like dimethyl sulfoxide (Me2SO) is important for cellular physiology.
• Solvents can alter enzyme activity by affecting the local microenvironment.

Purpose of the Study:

• To investigate the effects of dimethyl sulfoxide (Me2SO) and ethylene glycol on dog kidney (Na+ + K+)-ATPase activity.
• To elucidate the mechanism by which these solvents inhibit and modify enzyme kinetics.
• To understand how Me2SO influences different catalytic steps and substrate interactions.

Main Methods:

• Enzyme assays measuring (Na+ + K+)-ATPase, K+-nitrophenylphosphatase, and K+-acetylphosphatase activities.
• Kinetic analysis to determine inhibition patterns (uncompetitive, competitive, non-competitive) and kinetic parameters (Km, Vmax, Ki).
• Assessment of enzyme inactivation rates in the presence of Me2SO and specific inhibitors.

Main Results:

• Me2SO and ethylene glycol inhibited (Na+ + K+)-ATPase activity, with Me2SO showing uncompetitive inhibition towards ATP.
• Me2SO differentially affected K+-nitrophenylphosphatase activity, initially stimulating it at low concentrations and inhibiting it at higher concentrations.
• Me2SO altered kinetic parameters (Km, Ki) for various substrates and inhibitors, and increased enzyme inactivation rates by fluoride and beryllium.

Conclusions:

• Me2SO and ethylene glycol inhibit (Na+ + K+)-ATPase by increasing active site hydrophobicity, mimicking the E1P to E2P transition.
• These solvents stabilize enzyme intermediates (E2P), acting as dead-end inhibitors and affecting substrate/inhibitor binding.
• The findings provide mechanistic insights into enzyme regulation by solvent effects and active site properties.