Lüpfert C, Grell E, Pintschovius V, Apell H J, Cornelius F, Clarke R J
School of Chemistry, University of Sydney, Sydney NSW 2006, Australia.
Biophys J. 2001 Oct;81(4):2069-81. doi: 10.1016/S0006-3495(01)75856-0.
The kinetics of Na(+)-dependent phosphorylation of the Na(+),K(+)-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na(+)], and [Mg(2+)], pH 7.4, and 24 degrees C). The well-established effect of buffer composition on the E(2)-E(1) equilibrium was used as a tool to investigate the effect of the initial enzyme conformation on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E(2)) yielded a 1/tau value of 59 s(-1). Addition of MgCl(2) (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/tau to values of 129, 167, and 143 s(-1), respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation from the E(2) state to an E(1) or E(1)-like state. The results thus demonstrate conclusively that the E(2) --> E(1) transition does in fact limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E(2) --> E(1) transition under physiological conditions could be estimated to be approximately 65 s(-1) for pig kidney enzyme and 90 s(-1) for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (approximately 48 s(-1) and approximately 43 s(-1) for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the alpha(1) isoform the E(2) --> E(1) conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.
利用荧光标记RH421，通过停流技术研究了在饱和[ATP]、[Na⁺]、[Mg²⁺]、pH 7.4及24℃条件下，ATP对Na⁺,K⁺-ATP酶的Na⁺依赖性磷酸化动力学。利用缓冲液组成对E₂-E₁平衡的既定影响作为工具，研究初始酶构象对酶磷酸化速率的影响。将猪肾酶在25 mM组氨酸和0.1 mM EDTA溶液（有利于E₂的条件）中预孵育，得到的1/τ值为59 s⁻¹。向预孵育溶液中加入MgCl₂（5 mM）、NaCl（2 mM）或ATP（2 mM），导致1/τ分别增加到129、167和143 s⁻¹。这些增加可归因于磷酸化前酶构象平衡从E₂状态向E₁或类似E₁状态的转变。结果因此确凿地证明，E₂→E₁转变实际上限制了泵循环后续反应的速率。基于实验结果，猪肾酶在生理条件下E₂→E₁转变的速率常数估计约为65 s⁻¹，兔肾酶约为90 s⁻¹。考虑到其他部分反应的速率，计算机模拟表明这些值与从稳态测量计算得到的酶循环周转数（猪和兔分别约为48 s⁻¹和约43 s⁻¹）一致。因此，对于α₁同工型酶，E₂→E₁构象变化被证明是整个酶循环的主要速率决定步骤。
