Mutation Lys758 --> Ile of the sarcoplasmic reticulum Ca2+-ATPase enhances dephosphorylation of E2P and inhibits the E2 to E1Ca2 transition.
The highly conserved lysine residue Lys758 in the fifth stalk segment of the sarcoplasmic reticulum Ca2+-ATPase was substituted with either isoleucine or arginine by site-directed mutagenesis. The substitution with arginine was without significant effects on Ca2+-ATPase function, whereas multiple changes of functional characteristics were observed with the Lys758 --> Ile mutant. These included insensitivity of ATPase activity to the calcium ionophore A23187, an alkaline shift of the pH dependence of ATPase activity, reduced maximum molecular turnover rate and steady-state phosphorylation level, reduced apparent affinities for Ca2+ and inorganic phosphate, as well as increased sensitivity to inhibition by vanadate. Analysis of the partial reaction steps of the enzyme cycle traced these changes to two steps. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate (E2P) was increased, irrespective of variations of pH, K+, Ca2+, and dimethyl sulfoxide concentration. In addition, the rate of conversion of the dephosphoenzyme with low Ca2+ affinity (E2) to the Ca2+-bound form activated for phosphorylation (E1Ca2) was reduced in the mutant, and the ATP-induced rate enhancement of this step required higher ATP concentrations in the mutant compared with the wild type.
Sorensen
T
T
Department of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark. jpa@fi.aau.dk
Vilsen
B
B
Andersen
J P
JP
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001692:Biological Transport; D019556:COS Cells; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology; D007532:Isoleucine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011994:Recombinant Proteins; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D007476:Ionophores; D011994:Recombinant Proteins; D007532:Isoleucine; D000001:Calcimycin; D001120:Arginine; D000252:Calcium-Transporting ATPases; D008239:Lysine; D002118:Calcium