Functional consequences of mutations in the beta-strand sector of the Ca2(+)-ATPase of sarcoplasmic reticulum.
Kinetic studies of the phosphoenzyme intermediates of site-specific mutants were used to examine the role of Gly233 in the reaction mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase. When this glycine residue, which is highly conserved among cation-transporting ATPases, was replaced by valine, arginine, or glutamic acid, a complete loss of the ability to pump Ca2+ was observed. The mutant enzymes were able to form an ADP-sensitive phosphoenzyme intermediate (E1P) by reaction with ATP in the presence of Ca2+, but this intermediate decayed to the ADP-insensitive form (E2P) very slowly, relative to the wild-type enzyme. The mutant phosphoenzyme intermediate remained ADP-sensitive, even when phosphorylation from ATP was performed under conditions which permitted accumulation of the ADP-insensitive phosphoenzyme intermediate in the wild type. The mutants were also defective in their ability to form the ADP-insensitive phosphoenzyme intermediate by phosphorylation from inorganic phosphate. In addition, they displayed a higher affinity for Ca2+ and a lower cooperativity in Ca2+ binding than did the wild-type enzyme, as measured through the phosphorylation reaction with ATP. These findings can be rationalized either in terms of a parallel shift of E1 to E2 and E1P to E2P conformational equilibria toward the E1 and E1P forms, respectively, or in terms of destabilization of the phosphoryl-protein interaction in the E2P form. The roles of 7 other residues located in the vicinity of Gly233 were also examined by mutation. Although the side chains of these residues are potential Ca2+ ligands, their replacement did not affect the Ca2+ affinity of the enzyme, suggesting the lack of a role of this region of the peptide in formation of Ca2(+)-binding sites.
Andersen
J P
JP
Banting and Best Department of Medical Research, C. H. Best Institute, University of Toronto, Ontario, Canada.
Vilsen
B
B
Leberer
E
E
MacLennan
D H
DH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D007700:Kinetics; D009154:Mutation; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000596:Amino Acids; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium