Functional consequences of mutations of conserved amino acids in the beta-strand domain of the Ca2(+)-ATPase of sarcoplasmic reticulum.
The sequences Thr-Gly-Glu-Ser184 and Asp-Gln-Ser178 and individual residues Asp149, Asp157, and Asp162 in the sarcoplasmic reticulum Ca2(+)-ATPase are highly conserved throughout the family of cation-transporting ATPases. Mutant Thr181----Ala, Gly182----Ala, Glu183----Ala, and Glu183----Gln, created by in vitro mutagenesis, were devoid of Ca2+ transport activity. None of these mutations, however, affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+, indicating that the high affinity Ca2(+)-binding sites and the nucleotide-binding sites were intact. In each of these mutants, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive form (E2P) very slowly relative to the wild-type enzyme, whereas E2P decayed at a rate similar to that of the wild-type enzyme. Thus, the inability of the mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. These results suggest that Thr181, Gly182, and Glu183 play essential roles in the conformational change between E1P and E2P. Mutation of Ser184, Asp157, or Ser178 had little or no effect on either Ca2+ transport activity or expression. Mutations of Asp149, Asp162, and Gln177, however, were poorly expressed. Where expression could be measured, in mutations to Asp162 and Gln177, Ca2+ transport activity was essentially equivalent to that of the wild-type enzyme.
Clarke
D M
DM
Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, Ontario, Canada.
Loo
T W
TW
MacLennan
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003062:Codon / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009154:Mutation; D015345:Oligonucleotide Probes; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014162:Transfection
D003062:Codon; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D000252:Calcium-Transporting ATPases