Allosteric effect of ATP on Na+,K+-ATPase conformational kinetics

The kinetics of the E-2 -> E-1 conformational change of unphosphorylated Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was re-equilibrated in a solution containing 25 mM histidine and 0. 1 mM EDTA to stabilize the E-2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 MM NaCl and varying concentrations of Na(2)ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of >= 50 mu M, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E-2:E-2 -> E-2:E-1 -> E-1:E-1. In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E-2:E-2 -> E-2:E-1 and E-2:E-1 -> E-1:E-1 transitions were found to be 10.4 (+/- 1.0) and 0.49 (+/- 0.02) s(-1), respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 (+/- 0.3) s(-1). It was found that ATP acting at a high affinity site (K-d approximate to 0.25 mu M), stimulated the reverse reaction, E(1)ATP -> E(2)ATP, in addition to its known allosteric low affinity (K-d approximate to 71 mu M) stimulation of the forward reaction, E(2)ATP -> E(1)ATP.