Functional consequences of alterations to amino acids at the M5S5 boundary of the Ca(2+)-ATPase of sarcoplasmic reticulum. Mutation Tyr763-->Gly uncouples ATP hydrolysis from Ca2+ transport.
The roles of the hydrophobic side chains of residues Phe760, Ile761, Tyr763, Leu764, and Ile765 located at the M5S5 boundary of the Ca(2+)-ATPase of sarcoplasmic reticulum were analyzed by site-directed mutagenesis. Substitution of Tyr763 with glycine resulted in a new phenotypic variant of the Ca(2+)-ATPase that catalyzed a high rate of Ca(2+)-activated ATP hydrolysis without net accumulation of Ca2+ in the microsomal vesicles. The ATPase activity of the Tyr763-->Gly mutant displayed characteristics similar to the ATPase activity of the wild-type enzyme measured in the presence of calcium ionophore, and the mutant was able to form the ADP-insensitive phosphoenzyme intermediate. Mutants Phe760-->Gly, Ile761-->Gly, Leu764-->Gly, and Ile765-->Gly were able to accumulate Ca2+. In mutants Leu764-->Gly and Ile765-->Gly, the turnover rate was low due to inhibition of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate. On the other hand, mutant Leu764-->Lys dephosphorylated rapidly. Mutants Phe760-->Gly and Leu764-->Lys displayed apparent Ca2+ affinities that were reduced two and three orders of magnitude, respectively, relative to that of the wild-type.
Andersen
J P
JP
Danish Biomembrane Research Centre, University of Aarhus.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014443:Tyrosine / Q000378:metabolism*
D010710:Phosphates; D014443:Tyrosine; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; D005998:Glycine