The
kinetics of the E
2 ![](/images/entities/rarr.gif)
E
1 conformational change of unphosphorylated Na
+,K
+-ATPasewas investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24
![](/images/entities/deg.gif)
C). Theenzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize theE
2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl andvarying concentrations of Na
2ATP simultaneously, a fluorescence decrease was observed. In the absenceof ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations aftermixing of
![](/images/entities/ge.gif)
50
![](/images/entities/mgr.gif)
M, the fluorescence transient could be adequately fitted by a single exponential. On thebasis of the agreement between theoretical simulations and experimental traces, we propose that in theabsence of bound ATP the conformational transition occurs as a two step reversible process within aprotein dimer, E
2:E
2 ![](/images/entities/rarr.gif)
E
2:E
1 ![](/images/entities/rarr.gif)
E
1:E
1. In the presence of 130 mM NaCl, the sum of the forward andbac
kward rate constants for the E
2:E
2 ![](/images/entities/rarr.gif)
E
2:E
1 and E
2:E
1 ![](/images/entities/rarr.gif)
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 transitionoccurs in a single reversible step with the sum of its forward and bac
kward rate constants equal to 35.2(±0.3) s
-1. It was found that ATP acting at a high affinity site (
Kd ![](/images/entities/ap.gif)
0.25
![](/images/entities/mgr.gif)
M), stimulated the reversereaction, E
1ATP
![](/images/entities/rarr.gif)
E
2ATP, in addition to its
known allosteric low affinity (
Kd ![](/images/entities/ap.gif)
71
![](/images/entities/mgr.gif)
M) stimulation ofthe forward reaction, E
2ATP
![](/images/entities/rarr.gif)
E
1ATP.