Mutations of Arg198 in sarcoplasmic reticulum Ca2+-ATPase cause inhibition of hydrolysis of the phosphoenzyme intermediate formed from inorganic phosphate

Arg(198) of sarcoplasmic reticulum Ca2+-ATPase was substituted with lysine, glutamine, glutamic acid, alanine, and isoleucine by site-directed mutagenesis. Kinetic analysis was performed with microsomal membranes isolated from COS-I cells which were transfected,vith the mutated cDNAs. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was determined by first phosphorylating the Ca2+-ATPase with P-32(i) and then diluting the sample with non-radioactive P-i. This rate mas 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 R198K. The reduction in the rate of dephosphorylation was consistent with the observed decrease in the turnover rate of the Ca2+-ATPase accompanied by the steady-state accumulation of the ADP-insensitive phosphoenzyme formed from ATP. These results indicate that the positive charge and high hydrophilicity. of Arg(198) are critical for rapid hydrolysis of the ADP-insensitive phosphoenzyme. (C) 1999 Federation of European Biochemical Societies.