Functional consequences of alterations to Thr247, Pro248, Glu340, Asp813, Arg819, and Arg822 at the interfaces between domain P, M3, and L6-7 of sarcoplasmic reticulum Ca2+-ATPase -: Roles in Ca2+ interaction and phosphoenzyme processing

Point mutants with alterations to amino acid residues Thr(247), Pro(248), Glu(340), Asp(813), Arg(819), and Arg(822) of sarcoplasmic reticulum Ca2+-ATPase were analyzed by transient kinetic measurements. In the Ca2+-ATPase crystal structures, most of these residues participate in a hydrogen-bonding network between the phosphorylation domain (domain P), the third transmembrane helix (M3), and the cytoplasmic loop connecting the sixth and the seventh transmembrane helices (L6-7). In several of the mutants, a pronounced phosphorylation overshoot was observed upon reaction of the Ca2+-bound enzyme with ATP, because of accumulation of dephosphoenzyme at steady state. Mutations of Glu(340) and its partners, Thr(247) and Arg(822), in the bonding network markedly slowed the Ca2+ binding transition (E2-->E1-->Ca(2)E1) as well as Ca2+ dissociation from Ca2+ site II back toward the cytosol but did not affect the apparent affinity for vanadate. These mutations may have caused a slowing, in both directions, of the conformational change associated directly with Ca2+ interaction at Ca2+ site II. Because mutation of Asp(813) inhibited the Ca2+ binding transition, but not Ca2+ dissociation, and increased the apparent affinity for vanadate, the effect on the Ca2+ binding transition seems in this case to be exerted by slowing the E2-->E1 conformational change. Because the rate was not significantly enhanced by a 10-fold increase of the Ca2+ concentration, the slowing is not the consequence of reduced affinity of any pre-binding site for Ca2+. Furthermore, the mutations interfered in specific ways with the phosphoenzyme processing steps of the transport cycle; the transition from ADP-sensitive phosphoenzyme to ADP-insensitive phosphoenzyme (Ca(2)E1P-->E2P) was accelerated by mutations perturbing the interactions mediated by Glu(340) and Asp(813) and inhibited by mutation of Pro(248), and mutations of Thr(247) induced charge-specific changes of the rate of dephosphorylation of E2P.