Stopped-Flow Kinetic Investigations of Conformational Changes of Pig Kidney Na+,K+-ATPase

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Abstract
The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/τ1 ≈ 180 s-1 (saturating [ATP] and [Na+], pH 7.4 and 24 °C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 → E2P(Na+)3 + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 μM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (≥600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/τ1 showed half-saturation at a Na+ concentration of 8−10 mM with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high affinity ATP binding site determined from the ATP concentration dependence of 1/τ1 was 7.0 (±0.6) μM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (±17) μM and ≤ 28 s-1. At RH421 concentrations in the micromolar range, a decrease in the value of 1/τ1 is observed. On the basis of rapid quenched-flow measurements, this inhibition can be attributed to a reaction step subsequent to phosphorylation. This accounts for previously observed kinetic discrepancies between RH421 and BIPM.