AMINO-ACIDS ASN(796) AND THR(799) OF THE CA2-ATPASE OF SARCOPLASMIC-RETICULUM BIND CA2+ AT DIFFERENT SITES()

The Ca2+-binding properties of mutants Asn(798) --> Ala, Thr(799) --> Ala, and Glu(908) -->) Ala of the sarcoplasmic reticulum Ca2+-ATPase w ere analyzed in studies of CrATP-induced Ca2+ occlusion and of the Ca2 + dependencies of phosphorylation from ATP and P-i. The Asn(796) --> A la and Thr(799) --> Ala mutants were unable to occlude Ca2+, whereas t he Glu(908) --> Ala mutant was fully capable of occluding Ca2+. Mutant Asn(796) --> Ala was unable to form a phosphoenzyme from ATP, whereas mutants Thr(799), Ala and Glu(908) --> Ala phosphorylated from ATP wi th K-0.5 values for Ca2+ activation of 3 and 0.4 mM, respectively. In the Asn(796) --> Ala mutant, Ca2+ inhibited phosphorylation from P-i w ith a K-0.5 value of less than 10 mu M, whereas in mutants Thr(799) -- > Ala and Glu(908) --> Ala, the K-0.5 values for Ca2+ inhibition of ph osphorylation from P-i were much higher and similar to the K-0.5 value s observed for Ca2+ activation of phosphorylation from ATP. These data are consistent with a model in which the respective side chains of As n(796) and Thr(799) are assigned to two different Ca2+ sites being inv olved in Ca2+ occlusion, while the side chain of Glu(908) is excluded as a direct Ca2+ ligand in the Ca2+ occluded complex. The dephosphoryl ation of the ADP-insensitive E(2)P phosphoenzyme intermediate was bloc ked in the Asn(796) --> Ala mutant, suggesting the possibility that As n(796) participates in the countertransport of protons.