MUTATION LYS(758)-]ILE OF THE SARCOPLASMIC-RETICULUM CA2-ATPASE ENHANCES DEPHOSPHORYLATION OF E2P AND INHIBITS THE E-2 TO E1CA2 TRANSITION()

The highly conserved lysine residue Lys(758) in the fifth stalk segmen t of the sarcoplasmic reticulum Ca2+-ATPase was substituted with eithe r isoleucine or arginine by site-directed mutagenesis. The substitutio n with arginine was without significant effects on Ca2+-ATPase functio n, whereas multiple changes of functional characteristics were observe d with the Lys(758) --> Ile mutant. These included insensitivity of AT Pase activity to the calcium ionophore A23187, an alkaline shift of th e pH dependence of ATPase activity, reduced maximum molecular turnover rate and steady-state phosphorylation level, reduced apparent affinit ies 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 dephos phorylation of the ADP-insensitive phosphoenzyme intermediate (E2P) wa s increased, irrespective of variations of pH, K+, Ca2+, and dimethyl sulfoxide concentration. In addition, the rate of conversion of the de phosphoenzyme with low Ca2+ affinity (E-2) to the Ca2+-bound form acti vated for phosphorylation (E1Ca2) was reduced in the mutant, and the A TP-induced rate enhancement of this step required higher ATP concentra tions in the mutant compared with the wild type.