Allosteric Effect of ATP on Na+,K+-ATPase Conformational Kinetics
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- 作者：Ronald J. Clarke ; Hans-Jü ; rgen Apell ; Benjamin Y. Kong
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：June 12, 2007
- 年：2007
- 卷：46
- 期：23
- 页码：7034 - 7044
- 全文大小：142K
- 年卷期：v.46,no.23(June 12, 2007)
- ISSN：1520-4995
文摘
The kinetics of the E2 
E1 conformational change of unphosphorylated Na+,K+-ATPasewas investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 
C). Theenzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize theE2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl andvarying concentrations of Na2ATP simultaneously, a fluorescence decrease was observed. In the absenceof ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations aftermixing of 
50 
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, E2:E2 E2:E1 E1:E1. In the presence of 130 mM NaCl, the sum of the forward andbackward rate constants for the E2:E2 E2:E1 and E2:E1 E1:E1 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 backward rate constants equal to 35.2(±0.3) s-1. It was found that ATP acting at a high affinity site (Kd 
0.25 M), stimulated the reversereaction, E1ATP E2ATP, in addition to its known allosteric low affinity (Kd 71 M) stimulation ofthe forward reaction, E2ATP E1ATP.
E1 conformational change of unphosphorylated Na+,K+-ATPasewas investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 C). Theenzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize theE2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl andvarying concentrations of Na2ATP simultaneously, a fluorescence decrease was observed. In the absenceof ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations aftermixing of 50 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, E2:E2 E2:E1 E1:E1. In the presence of 130 mM NaCl, the sum of the forward andbackward rate constants for the E2:E2 E2:E1 and E2:E1 E1:E1 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 backward rate constants equal to 35.2(±0.3) s-1. It was found that ATP acting at a high affinity site (Kd 0.25 M), stimulated the reversereaction, E1ATP E2ATP, in addition to its known allosteric low affinity (Kd 71 M) stimulation ofthe forward reaction, E2ATP E1ATP.