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

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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.