Andersen J P, Vilsen B, Leberer E, MacLennan D H
Banting and Best Department of Medical Research, C. H. Best Institute, University of Toronto, Ontario, Canada.
J Biol Chem. 1989 Dec 15;264(35):21018-23.
Kinetic studies of the phosphoenzyme intermediates of site-specific mutants were used to examine the role of Gly233 in the reaction mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase. When this glycine residue, which is highly conserved among cation-transporting ATPases, was replaced by valine, arginine, or glutamic acid, a complete loss of the ability to pump Ca2+ was observed. The mutant enzymes were able to form an ADP-sensitive phosphoenzyme intermediate (E1P) by reaction with ATP in the presence of Ca2+, but this intermediate decayed to the ADP-insensitive form (E2P) very slowly, relative to the wild-type enzyme. The mutant phosphoenzyme intermediate remained ADP-sensitive, even when phosphorylation from ATP was performed under conditions which permitted accumulation of the ADP-insensitive phosphoenzyme intermediate in the wild type. The mutants were also defective in their ability to form the ADP-insensitive phosphoenzyme intermediate by phosphorylation from inorganic phosphate. In addition, they displayed a higher affinity for Ca2+ and a lower cooperativity in Ca2+ binding than did the wild-type enzyme, as measured through the phosphorylation reaction with ATP. These findings can be rationalized either in terms of a parallel shift of E1 to E2 and E1P to E2P conformational equilibria toward the E1 and E1P forms, respectively, or in terms of destabilization of the phosphoryl-protein interaction in the E2P form. The roles of 7 other residues located in the vicinity of Gly233 were also examined by mutation. Although the side chains of these residues are potential Ca2+ ligands, their replacement did not affect the Ca2+ affinity of the enzyme, suggesting the lack of a role of this region of the peptide in formation of Ca2(+)-binding sites.
通过对位点特异性突变体的磷酸化酶中间体进行动力学研究，以考察甘氨酸233在肌浆网Ca2 + -ATP酶反应机制中的作用。当这个在阳离子转运ATP酶中高度保守的甘氨酸残基被缬氨酸、精氨酸或谷氨酸取代时，观察到其泵浦Ca2 + 的能力完全丧失。突变酶能够在Ca2 + 存在的情况下通过与ATP反应形成ADP敏感的磷酸化酶中间体（E1P），但相对于野生型酶，这种中间体向ADP不敏感形式（E2P）的衰减非常缓慢。即使在允许野生型中积累ADP不敏感的磷酸化酶中间体的条件下进行ATP磷酸化时，突变的磷酸化酶中间体仍对ADP敏感。这些突变体通过无机磷酸磷酸化形成ADP不敏感的磷酸化酶中间体的能力也存在缺陷。此外，通过与ATP的磷酸化反应测定，它们对Ca2 + 的亲和力更高，并且在Ca2 + 结合方面的协同性比野生型酶更低。这些发现可以分别根据E1到E2以及E1P到E2P构象平衡分别向E1和E1P形式的平行移动来解释，或者根据E2P形式中磷蛋白相互作用的不稳定来解释。还通过突变研究了位于甘氨酸233附近的其他7个残基的作用。尽管这些残基的侧链是潜在的Ca2 + 配体，但它们的取代并不影响酶对Ca2 + 的亲和力，这表明该肽段的这一区域在形成Ca2 + 结合位点中缺乏作用。
