Buxbaum E, Schoner W
Institut für Biochemie und Endokrinologie, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Eur J Biochem. 1991 Jan 30;195(2):407-19. doi: 10.1111/j.1432-1033.1991.tb15720.x.
Tetrammine cobalt(III) phosphate [Co(NH3)4PO4] inactivates Na+/K(+)-ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1): Co(NH3)4PO4 + E2 Kd in equilibrium E2.Co(NH3)4PO4k2----E'2.Co(NH3)4PO4. The inactivation rate constant k2 for the formation of a stable E'2.Co(NH3)4PO4 at 37 degrees C was 0.057 min-1; the dissociation constant, Kd = 300 microM. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5'-[beta, gamma-methylene]triphosphate competed with Co(NH3)4PO4 for its binding site with Ks = 0.41 mM and 5 mM, respectively. MgPO4 competed with Co(NH3)4PO4 linearly, with Ks = 50 microM, as did phosphate (Ks = 16 mM) and Mg2+ (Ks = 160 microM). It is concluded that the MgPO4 analogue binds to the MgPO4-binding subsite of the low-affinity ATP-binding site (of the E2 conformation). Also, Na+ (Ks = 860 microM) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na+ against the inactivation by Co(NH3)4PO4, apparent affinities of K+ for the free enzyme of 41 microM, and for the E.Co(NH3)4PO4 complex of 720 microM, were calculated. Binding of Co(NH3)4PO4 to the enzyme inactivated Na+/K(+)-ATPase and K(+)-activated phosphatase, and, moreover, prevented the occlusion of 86Rb+; however, the activity of the Na(+)-ATPase, the phosphorylation capacity of the high-affinity ATP-binding site and the ATP/ADP-exchange reaction remained unchanged. With Co(NH3)432PO4 a binding capacity of 135 pmol unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with Co(NH3)432PO4 or the 32P-labelled tetramminecobalt ATP ([gamma-32P]Co(NH3)4ATP) at the low-affinity ATP-binding site, allowed (independent of the purity of the Na+/K(+)-ATPase preparation) a further incorporation of radioactivity from 32P-labelled tetraaquachromium(III) ATP ([gamma-32P]CrATP) to the high-affinity ATP-binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K(+)-ATPase by [gamma-32P]CrATP prevented the binding of Co(NH3)4 32PO4 or [gamma-32P]Co(NH3)4ATP to the enzyme. [gamma-32P]CO(NH3)4ATP and Co(NH3)432PO4 are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (alpha,beta)2-diprotomer, which change their interactions during the Na+/K(+)-pumping process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action.
四氨合钴（III）磷酸盐[Co(NH₃)₄PO₄]可使处于E2构象状态的Na⁺/K⁺-ATP酶失活，其失活程度取决于时间和浓度，符合方程式（1）：Co(NH₃)₄PO₄ + E2 ⇌ Kd（平衡）E2·Co(NH₃)₄PO₄ →k₂ E'2·Co(NH₃)₄PO₄。在37℃时，形成稳定的E'2·Co(NH₃)₄PO₄的失活速率常数k₂为0.057 min⁻¹；解离常数Kd = 300 μM。失活过程的活化能为149 kJ/mol。ATP和不可裂解的腺苷5'-[β,γ-亚甲基]三磷酸分别以Ks = 0.41 mM和5 mM与Co(NH₃)₄PO₄竞争其结合位点。MgPO₄与Co(NH₃)₄PO₄呈线性竞争，Ks = 50 μM，磷酸盐（Ks = 16 mM）和Mg²⁺（Ks = 160 μM）也是如此。可以得出结论，MgPO₄类似物与低亲和力ATP结合位点（E2构象）的MgPO₄结合亚位点结合。此外，Na⁺（Ks = 860 μM）以竞争性方式保护酶不被失活。根据Na⁺对Co(NH₃)₄PO₄失活的交叉（斜率和截距呈线性）非竞争性效应，计算出K⁺对游离酶的表观亲和力为41 μM，对E·Co(NH₃)₄PO₄复合物的表观亲和力为720 μM。Co(NH₃)₄PO₄与酶的结合使Na⁺/K⁺-ATP酶和K⁺激活的磷酸酶失活，此外，还阻止了⁸⁶Rb⁺的封闭；然而，Na⁺-ATP酶的活性、高亲和力ATP结合位点的磷酸化能力以及ATP/ADP交换反应保持不变。发现Co(NH₃)₄³²PO₄的结合容量为135 pmol/单位酶。用Co(NH₃)₄³²PO₄或³²P标记的四氨合钴ATP（[γ-³²P]Co(NH₃)₄ATP）在低亲和力ATP结合位点使酶磷酸化并完全失活，（与Na⁺/K⁺-ATP酶制剂的纯度无关）允许³²P标记的四水合铬（III）ATP（[γ-³²P]CrATP）的放射性进一步掺入高亲和力ATP结合位点，且磷酸化能力不变。然而，[γ-³²P]CrATP对Na⁺/K⁺-ATP酶的失活和磷酸化阻止了Co(NH₃)₄³²PO₄或[γ-³²P]Co(NH₃)₄ATP与酶的结合。[γ-³²P]Co(NH₃)₄ATP和Co(NH₃)₄³²PO₄相互排斥。这些数据与（α,β）₂二聚体中催化亚基协同作用的假设一致，这些亚基在Na⁺/K⁺泵浦过程中改变它们的相互作用。我们的发现似乎不支持酶作用的对称Repke和Stein模型。
