Abstract
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.
Highlights
Thought to comprise 10 transmembrane helices M1-M10 [2, 5, 6]
Ionophore Sensitivity and Ca2ϩ Dependence of ATPase Activity of Mutant Lys758 3 Ile—Steady-state rates of ATP hydrolysis were determined for the wild type and mutant Lys758 3 Ile at various Ca2ϩ concentrations at pH 7.0, in the presence and absence of the calcium ionophore A23187 (Fig. 2)
By allowing passive efflux of Ca2ϩ accumulated inside the microsomal vesicles the calcium ionophore increases the ATPase activity of the wild type 2–3-fold, due to relief of the “back inhibition” of the E1PCa2 to E2P transformation imposed by a high lumenal Ca2ϩ concentration
Summary
S1-S5, stalk segments connecting M1-M5 with the cytoplasmic domains; E1Ca2, Ca2ϩ-ATPase conformation with two calcium ions bound at cytoplasmically facing high-affinity sites; E2, Ca2ϩ-ATPase conformation with low Ca2ϩ affinity; E1PCa2, phosphorylated Ca2ϩ-ATPase conformation containing occluded Ca2ϩ and being sensitive to ADP; E2P, phosphorylated Ca2ϩ-ATPase conformation insensitive to ADP; M1-M10, putative transmembrane segments numbered from the NH2-terminal end of the peptide chain; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)-propanesulfonic acid; TES, Ntris[hydroxymethyl]methyl-2-aminoethanesulfonic acid. The stalk segments S4 and S5 that physically link the largest cytoplasmic loop containing the phosphorylated aspartic acid residue (Asp351) to transmembrane segments M4 and M5 are logical candidates for structural elements being involved in the intramolecular signaling between the membrane domain and the catalytic site, and several of the residues shown to be crucial to the E1PCa2 to E2P transition are located in the S4 segment [3, 12, 14, 15]. Mutation Lys758 Ile results in multiple changes of phenotypic characteristics, which can be traced to an increased rate of dephosphorylation of E2P and a decreased rate of the Ca2ϩ-binding E2 to E1Ca2 transition. These changes have not previously been observed with a point mutant of the Ca2ϩ-ATPase
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
