Mutations of Arg198 in sarcoplasmic reticulum Ca2+-ATPase cause inhibition of hydrolysis of the phosphoenzyme intermediate formed from inorganic phosphate.
Arg198 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-1 cells which were transfected with the mutated cDNAs. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was determined by first phosphorylating the Ca2+-ATPase with 32Pi and then diluting the sample with non-radioactive Pi. This rate was reduced substantially in the mutant R198Q, more strongly in the mutants R198A and R1981, 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 Arg198 are critical for rapid hydrolysis of the ADP-insensitive phosphoenzyme.
Daiho
T
T
Department of Biochemistry, Asahikawa Medical College, Japan.
Suzuki
H
H
Yamasaki
K
K
Saino
T
T
Kanazawa
T
T
D000244:Adenosine Diphosphate / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics*; D015153:Blotting, Western; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014162:Transfection
D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D000252:Calcium-Transporting ATPases; D011188:Potassium