Inesi G, Lewis D, Murphy A J
J Biol Chem. 1984 Jan 25;259(2):996-1003.
The phosphorylation of sarcoplasmic reticulum ATPase with Pi in the absence of Ca2+ was studied by equilibrium and kinetic experimentation. The combination of these measurements was then subjected to analysis without assumptions on the stoichiometry of the reactive sites. The analysis indicates that the species undergoing covalent interaction is the tertiary complex E X Pi X Mg formed by independent interaction of the two ligands with the enzyme. The binding constant of Pi or Mg2+ to either free or partially associated enzyme is approximately equal to 10(2) M-1, and no significant synergistic effect is produced by one ligand on the binding of the other; the equilibrium constant (Keq) for the covalent reaction E X Pi X Mg E-P X Mg is approximately equal to 16, with kphosph = 53 s-1, and khyd = 3-4 s-1 (25 degrees C, pH 6.0, no K+). The phosphorylation reaction of sarcoplasmic reticulum ATPase with Pi is highly H+ dependent. Such a pH dependence involves the affinity of enzyme for different ionization states of Pi, as well as protonation of two protein residues per enzyme unit in order to obtain optimal phosphorylation. The experimental data can then be fitted satisfactorily assuming pK values of 5.7 and 8.5 for the two residues in the nonphosphorylated enzyme (changing to 7.7 for one of the two residues, following phosphorylation) and values of 50.0 and 0.58 for the equilibrium constants of the H2(E X HPO4) in equilibrium with H(E-PO3) + H2O and H(E X HPO4) in equilibrium with E-PO3 + H2O reactions, respectively. In addition to the interdependence of H+ and phosphorylation sites, an interdependence of Ca2+ and phosphorylation sites is revealed by total inhibition of the Pi reaction when two high affinity calcium sites per enzyme unit are occupied by calcium. Conversely, occupancy of the phosphate site by vanadate (a stable transition state analogue of phosphate) inhibits high affinity calcium binding. The known binding competition between the two cations and their opposite effects on the phosphorylation reaction suggest that interdependence of phosphorylation site, H+ sites, and Ca2+ sites is a basic mechanistic feature of enzyme catalysis and cation transport.
通过平衡和动力学实验研究了在无Ca2+情况下肌浆网ATP酶与Pi的磷酸化作用。然后，在不假设反应位点化学计量的情况下，对这些测量结果进行分析。分析表明，发生共价相互作用的物质是由两个配体与酶独立相互作用形成的三级复合物E X Pi X Mg。Pi或Mg2+与游离或部分结合的酶的结合常数约为10(2) M-1，一个配体对另一个配体的结合没有显著的协同效应；共价反应E X Pi X Mg E-P X Mg的平衡常数(Keq)约为16，kphosph = 53 s-1，khyd = 3 - 4 s-1(25℃，pH 6.0，无K+)。肌浆网ATP酶与Pi的磷酸化反应高度依赖于H+。这种pH依赖性涉及酶对Pi不同电离状态的亲和力，以及每个酶单位中两个蛋白质残基的质子化，以获得最佳磷酸化。假设非磷酸化酶中两个残基的pK值分别为5.7和8.5(磷酸化后两个残基中的一个变为7.7)，以及H2(E X HPO4)与H(E-PO3) + H2O平衡和H(E X HPO4)与E-PO3 + H2O反应平衡常数分别为50.0和0.58，则实验数据可以得到满意的拟合。除了H+和磷酸化位点的相互依赖性外，当每个酶单位的两个高亲和力钙位点被钙占据时，Pi反应完全被抑制，这揭示了Ca2+和磷酸化位点的相互依赖性。相反，钒酸盐(磷酸盐的稳定过渡态类似物)占据磷酸位点会抑制高亲和力钙结合。两种阳离子之间已知的结合竞争及其对磷酸化反应的相反影响表明，磷酸化位点、H+位点和Ca2+位点的相互依赖性是酶催化和阳离子转运的基本机制特征。
