Kinetic Studies on Na+/K+-ATPase and Inhibition of Na+/K+-ATPase by ATP

Abstract

Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na+/K+-ATPase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (δrHm) but also the kinetic data (Km and Vmax). At 310.15 K and pH 7.4, the molar reaction enthalpy (δrHm) was measured as -40.514 ± 0.9 kJ mol−1. The Michaelis constant (Km) was determined to be 0.479 ± 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na+/K+-ATPase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a Km value of about 0.5 mM and free ATP was a competitive inhibitor with a Ki value of 0.253 mM.