FUNCTIONAL CONSEQUENCES OF ALTERATIONS TO AMINO-ACIDS AT THE M5S5 BOUNDARY OF THE CA2+-ATPASE OF SARCOPLASMIC-RETICULUM - MUTATION TYR763-]GLY UNCOUPLES ATP HYDROLYSIS FROM CA2+ TRANSPORT

The roles of the hydrophobic side chains of residues Phe(760), Ile(761), Tyr(763), Leu(764), and Ile(765) located at the M5S5 boundary of the Ca2+-ATPase of sarcoplasmic reticulum were analyzed by site-directed mutagenesis. Substitution of Tyr(763) with glycine resulted in a new phenotypic variant of the Ca2+-ATPase that catalyzed a high rate of Ca2+-activated ATP hydrolysis without net accumulation of Ca2+ in the microsomal vesicles. The ATPase activity of the Tyr(763), Gly mutant displayed characteristics similar to the ATPase activity of the mild-type enzyme measured in the presence of calcium ionophore, and the mutant was able to form the ADP-insensitive phosphoenzyme intermediate. Mutants Phe(760) --> Gly, Ile(761) --> Gly, Leu(764) --> Gly, and Ile(765) --> Gly were able to accumulate Ca2+. In mutants Leu(764) --> Gly and Ile(765) --> Gly, the turnover rate was low due to inhibition of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate. On the other hand, mutant Leu(764) --> Lys dephosphorylated rapidly. Mutants Phe(760) --> Gly and Leu(764) --> Lys displayed apparent Ca2+ affinities that were reduced two and three orders of magnitude, respectively, relative to that of the wild-type.