Structure and function of the catalytic site of sarcoplasmic reticulum Ca^<2+>-ATPase.

采用定点诱变的方法研究了Arg^<198>在肌浆网Ca^<2+>- atp酶中的功能作用，将Arg^<198>替换为赖氨酸、谷氨酰胺、谷氨酸、丙氨酸和异亮氨酸。将突变的cdna转染到COS-1细胞中，用从转染细胞中分离的微粒体膜进行动力学分析。突变体R198K的周转率与野生型基本相同，而突变体R198Q、R198A和R198I的周转率则相差很大。比野生型低。突变体R198E的周转率进一步降低。突变体R198K和野生型在稳态K^+存在下由ATP形成的磷酸化酶几乎完全对adp敏感。相比之下，adp不敏感的磷酸酶在突变体R198Q中积累了相当程度的积累，在突变体R198A和R198I中积累的程度更大，在突变体R198E中积累的程度最大。首先在没有K^+的情况下，用^<32>Pi磷酸化Ca^<2+>- atp酶，然后用含有非放射性Pi和KCl（或用LiCl代替KCl）的溶液稀释样品，以此来测定adp不敏感磷酸化酶的脱磷酸化时间过程。在存在或不存在K^+的情况下，突变体R198Q的去磷酸化率显著降低，突变体R198A和R198I的去磷酸化率更低，突变体R198E的去磷酸化率最高，但R198K的去磷酸化率要低得多。这些结果表明，Arg^<198>的正电荷和高亲水性是adp不敏感磷酸酶快速水解的关键。

The functional role of Arg^<198> in sarcoplasmic reticulum Ca^<2+>-ATPase was investigated by site-directed mutagenesis, in which Arg^<198> was substituted with lysine, glutamine, glutamic acid, alanine, and isoleucine. Mutated cDNAs were transfected into COS-1 cells, and kinetic analysis was performed with microsomal membranes isolated from the transfected cells. The turnover rate of the mutant R198K was almost the same as that of the wild type, whereas the turnover rates of the mutants R198Q, R198A, and R198I were substantially. lower than that of the wild type. The turnover rate was further reduced in the mutant R198E.The phosphoenzyme formed from ATP in the presence of K^+ at steady state was almost completely ADP-sensitive in the mutant R198K as well as in the wild type. In contrast, the ADP-insensitive phosphoenzyme accumulated to a considerable extent in the mutant R198Q, to a larger extent in the mutants R198A and R198I, and to the largest extent in the mutant R198E.The time course of dephosphorylation of the ADP-insensitive phosphoenzyme was determined by first phosphorylating the Ca^<2+>-ATPase with ^<32>Pi in the absence of K^+ and then diluting the sample with a solution containing nonradioactive Pi and KCl (or LiCl in place of KCl). The rate of dephosphorylation in the presence or absence of K^+ was reduced substantially in the mutant R198Q, more strongly in the mutants R198A and R198I, and most strongly in the mutant R198E, but to a much lesser extent in the R198K.These results indicate that the positive charge and high hydrophilicity of Arg^<198> are critical for rapid hydrolysis of the ADP-insensitive phosphoenzyme.