Jencks W P, Yang T, Peisach D, Myung J
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Biochemistry. 1993 Jul 13;32(27):7030-4. doi: 10.1021/bi00078a031.
The calcium-transporting ATPase of sarcoplasmic reticulum is known to bind two Ca2+ ions from the cytoplasm to the free enzyme and two Ca2+ ions from the lumen to the phosphoenzyme. The concentration of phosphoenzyme formed at equilibrium from Pi and Mg2+ increases with increasing concentration of calcium in the lumen, which binds to the phosphoenzyme to form Ca2.E approximately P.Mg. However, at subsaturating concentrations of Mg2+ increasing the concentration of lumenal Ca2+ does not drive phosphoenzyme formation to completion. The maximal levels of phosphoenzyme that are formed at saturating concentrations of lumenal Ca2+ increase with increasing concentrations of Mg2+. This result requires that Ca2+ can bind to low-affinity lumenal sites on both the free enzyme and the phosphoenzyme, as well as to the high-affinity cytoplasmic calcium-binding sites. If there were no lumenal binding sites for Ca2+ on the free enzyme, high concentrations of lumenal Ca2+ would convert all of the enzyme to the same maximal concentration of Ca2.E approximately P.Mg at subsaturating concentrations of Mg2+ and Pi. We conclude that there are two low-affinity lumenal sites as well as two high-affinity cytoplasmic sites for Ca2+ on the free enzyme. Phosphorylation by ATP results in translocation of Ca2+ from the high-affinity to the low-affinity sites.
已知肌质网的钙转运ATP酶可从细胞质中结合两个Ca2+离子到游离酶上，并从管腔中结合两个Ca2+离子到磷酸化酶上。由Pi和Mg2+在平衡时形成的磷酸化酶浓度随着管腔中钙浓度的增加而增加，钙与磷酸化酶结合形成Ca2.E约P.Mg。然而，在Mg2+亚饱和浓度下，增加管腔Ca2+浓度并不能使磷酸化酶形成完全。在管腔Ca2+饱和浓度下形成的磷酸化酶最大水平随着Mg2+浓度的增加而增加。这一结果表明Ca2+可以结合到游离酶和磷酸化酶上的低亲和力管腔位点，以及高亲和力的细胞质钙结合位点。如果游离酶上没有Ca2+的管腔结合位点，在Mg2+和Pi亚饱和浓度下，高浓度的管腔Ca2+会将所有酶转化为相同的最大浓度的Ca2.E约P.Mg。我们得出结论，游离酶上有两个低亲和力的管腔位点以及两个高亲和力的细胞质Ca2+位点。ATP磷酸化导致Ca2+从高亲和力位点转移到低亲和力位点。
