Amino acids Asn796 and Thr799 of the Ca(2+)-ATPase of sarcoplasmic reticulum bind Ca2+ at different sites.
The Ca(2+)-binding properties of mutants Asn796-->Ala, Thr799-->Ala, and Glu908-->Ala of the sarcoplasmic reticulum Ca(2+)-ATPase were analyzed in studies of CrATP-induced Ca2+ occlusion and of the Ca2+ dependencies of phosphorylation from ATP and P(i). The Asn796-->Ala and Thr799-->Ala mutants were unable to occlude Ca2+, whereas the Glu908-->Ala mutant was fully capable of occluding Ca2+. Mutant Asn796-->Ala was unable to form a phosphoenzyme from ATP, whereas mutants Thr799-->Ala and Glu908-->Ala phosphorylated from ATP with K0.5 values for Ca2+ activation of 3 and 0.4 mM, respectively. In the Asn796-->Ala mutant, Ca2+ inhibited phosphorylation from P(i) with a K0.5 value of less than 10 microM, whereas in mutants Thr799-->Ala and Glu908-->Ala, the K0.5 values for Ca2+ inhibition of phosphorylation from P(i) were much higher and similar to the K0.5 values observed for Ca2+ activation of phosphorylation from ATP. These data are consistent with a model in which the respective side chains of Asn796 and Thr799 are assigned to two different Ca2+ sites being involved in Ca2+ occlusion, while the side chain of Glu908 is excluded as a direct Ca2+ ligand in the Ca2+ occluded complex. The dephosphorylation of the ADP-insensitive E2P phosphoenzyme intermediate was blocked in the Asn796-->Ala mutant, suggesting the possibility that Asn796 participates in the countertransport of protons.
Andersen
J P
JP
Danish Biomembrane Research Centre, University of Aarhus.
Vilsen
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013912:Threonine; D014162:Transfection
D013912:Threonine; D001216:Asparagine; D000252:Calcium-Transporting ATPases; D002118:Calcium