Scheiner-Bobis G, Fahlbusch K, Schoner W
Institut für Biochemie und Endokrinologie, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Eur J Biochem. 1987 Oct 1;168(1):123-31. doi: 10.1111/j.1432-1033.1987.tb13396.x.
The MgATP complex analogue cobalt-tetrammine-ATP [Co(NH3)4ATP] inactivates (Na+ + K+)-ATPase at 37 degrees C slowly in the absence of univalent cations. This inactivation occurs concomitantly with incorporation of radioactivity from [alpha-32P]Co(NH3)4ATP and from [gamma-32P]Co(NH3)4ATP into the alpha subunit. The kinetics of inactivation are consistent with the formation of a dissociable complex of Co(NH3)4ATP with the enzyme (E) followed by the phosphorylation of the enzyme: (Formula: see text). The dissociation constant of the enzyme-MgATP analogue complex at 37 degrees C is Kd = 500 microM, the inactivation rate constant k2 = 0.05 min-1. ATP protects the enzyme against the inactivation by Co(NH3)4ATP due to binding at a site from which it dissociates with a Kd of 360 microM. It is concluded, therefore, that Co(NH3)4ATP binds to the low-affinity ATP binding site of the E2 conformational state. K+, Na+ and Mg2+ protect the enzyme against the inactivation by Co(NH3)4ATP. Whilst Na+ or Mg2+ decrease the inactivation rate constant k2, K+ exerts its protective effect by increasing the dissociation constant of the enzyme.Co(NH3)4ATP complex. The Co(NH3)4ATP-inactivated (Na+ + K+)-ATPase, in contrast to the non-inactivated enzyme, incorporates [3H]ouabain. This indicates that the Co(NH3)4ATP-inactivated enzyme is stabilized in the E2 conformational state. Despite the inactivation of (Na+ + K+)-ATPase by Co(NH3)4ATP from the low-affinity ATP binding site, there is no change in the capacity of the high-affinity ATP binding site (Kd = 0.9 microM) nor of its capability to phosphorylate the enzyme Na+-dependently. Since (Na+ + K+)-ATPase is phosphorylated Na+-dependently from the high-affinity ATP binding site although the catalytic cycle is arrested in the E2 conformational state by specific modification of the low-affinity ATP binding site, it is concluded that both ATP binding sites coexist at the same time in the working sodium pump. This demonstration of interacting catalytic subunits in the E1 and E2 conformational states excludes the proposal that a single catalytic subunit catalyzes (Na+ + K+)-transport.
在没有单价阳离子的情况下，MgATP复合类似物四氨合钴 - ATP [Co(NH₃)₄ATP] 在37℃时会缓慢使（Na⁺ + K⁺）-ATP酶失活。这种失活伴随着放射性从[α - ³²P]Co(NH₃)₄ATP和[γ - ³²P]Co(NH₃)₄ATP掺入α亚基。失活动力学与Co(NH₃)₄ATP与酶（E）形成可解离复合物随后酶的磷酸化过程一致：（公式：见原文）。在37℃时，酶 - MgATP类似物复合物的解离常数Kd = 500μM，失活速率常数k₂ = 0.05 min⁻¹。ATP可保护酶免受Co(NH₃)₄ATP导致的失活，因为它在一个位点结合，其解离常数Kd为360μM。因此可以得出结论，Co(NH₃)₄ATP与E₂构象状态的低亲和力ATP结合位点结合。K⁺、Na⁺和Mg²⁺可保护酶免受Co(NH₃)₄ATP导致的失活。虽然Na⁺或Mg²⁺会降低失活速率常数k₂，但K⁺通过增加酶 - Co(NH₃)₄ATP复合物的解离常数发挥其保护作用。与未失活的酶相比，Co(NH₃)₄ATP失活的（Na⁺ + K⁺）-ATP酶会掺入[³H]哇巴因。这表明Co(NH₃)₄ATP失活的酶在E₂构象状态下是稳定的。尽管Co(NH₃)₄ATP从低亲和力ATP结合位点使（Na⁺ + K⁺）-ATP酶失活，但高亲和力ATP结合位点（Kd = 0.9μM）的容量及其依赖Na⁺使酶磷酸化的能力均未改变。由于（Na⁺ + K⁺）-ATP酶从高亲和力ATP结合位点依赖Na⁺进行磷酸化，尽管催化循环通过低亲和力ATP结合位点的特异性修饰在E₂构象状态下被阻断，但可以得出结论，两个ATP结合位点在工作的钠泵中同时存在。E₁和E₂构象状态下相互作用的催化亚基的这一证明排除了单个催化亚基催化（Na⁺ + K⁺）转运的提议。
