Properties of the Conversion of an Enzyme-ATP Complex to a Phosphorylated Intermediate in the Reaction of Na⁺-K⁺-dependent ATPase

Abstract

Previously, we proposed the following reaction machanism for the transport ATPase [EC 3. 6. 1. 3] reaction in the presence of high concentrations of Mg²⁺ and Na⁺:
E+ATP_??_E₁ATP→E₂ATP_??_→E+ADP+P₁. step 1 2 3 4
Some kinetic and thermodynamic properties of steps 3 and 4 were investigated, and the following results were obtained.
1. When the reaction was started by adding ATP to the enzyme in the presence of 50mM Na⁺ and 0.5mM K⁺ or in the presence of 50mM Na⁺ and 0.5mM Rb⁺, the amount of_??_increased with time and maintained a constant level after reaching a maximum. We could not observe the initial burst of EP formation, which was observed by Post et al. in the presence of 8mM Na⁺ and 0.01mM Rb⁺.
2. The existence of quasi-equilibrium between E₂ATP and_??_in the presence of low concentrations of Na⁺ was suggested by the fact that the values of the reciprocal of the equilibrium constant, K₃, of step 3 obtained by the following three methods were almost the same. a) The value of 1+K3 was estimated from the ratio of v₀/[EP] to k_d, where v₀ is the rate of ATP hydrolysis in the steady state, [EP] the concentration of EP, and kd the first-order rate constant of EP disappearance after stopping EP formation. b) This value was also calculated from the ratio of the amount of P_i liberated to that of decrease in EP after stopping EP formation. c) The value of K₃ was also calculated from the initial rapid decrease in EP on adding K⁺ and EDTA, assuming that the rapid decrease was due to a shift of the equilibrium toward E2ATP on adding K⁺. For example, the value of K3 with 10mM NaCl and