Mutational analysis of Glu771 of the Ca(2+)-ATPase of sarcoplasmic reticulum. Effect of positive charge on dephosphorylation.
The glutamic acid residue Glu771 in the fifth transmembrane segment M5 of the Ca(2+)-ATPase of rabbit fast twitch muscle sarcoplasmic reticulum was substituted with lysine, alanine, and glycine by site-directed mutagenesis. Mutant Glu771-->Lys was unable to occlude Ca2+, and Ca2+ did not inhibit phosphorylation from P(i) or activate phosphorylation from ATP of this mutant. Mutants Glu771-->Ala and Glu771-->Gly were likewise unable to occlude Ca2+, but Ca2+ in the millimolar concentration range activated phosphorylation from ATP and inhibited phosphorylation from P(i) of these mutants. The dephosphorylation of the ADP-insensitive E2P phosphoenzyme intermediate of mutants Glu771-->Ala and Glu771-->Gly was found to be blocked, whereas the dephosphorylation proceeded rapidly for mutant Glu771-->Lys. This finding suggests a role of the positive charge of the lysine in induction of dephosphorylation, supporting the hypothesis that the side chain of Glu771 participates in the countertransport of two protons per Ca(2+)-ATPase cycle.
Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus.
J P
JP
Andersen
D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018698:Glutamic Acid / Q000502:physiology*; D008861:Microsomes / Q000378:metabolism; D018656:Muscle Fibers, Fast-Twitch / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology*; D010766:Phosphorylation; D011522:Protons; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014162:Transfection
D011522:Protons; D018698:Glutamic Acid; D000252:Calcium-Transporting ATPases