The role of the Ca2+ binding ligand Asn879 in the function of the plasma membrane Ca2+ pump.
Asn879 in the transmembrane segment M6 of the plasma membrane Ca(2+) pump (PMCA human isoform 4xb) has been proposed to coordinate Ca(2+) at the transport site through its carboxylate. This idea agrees with the fact that this Asn is conserved in other Ca(2+)-ATPases but is replaced by Asp, Glu, and other residues in closely related 2P-type ATPases of different ionic specificity. Previous mutagenesis studies have shown that the substitution of Ala for Asn abolishes the activity of the enzyme (Adebayo et al., 1995; Guerini et al., 1996). We have constructed a mutant PMCA in which the Asn879 was substituted by Asp. The mutant protein was expressed in Saccharomyces cerevisiae, solubilized and purified by calmodulin affinity chromatography. The Asn879Asp PMCA mutant exhibited about 30% of the wild type Ca(2+)-dependent ATPase activity and only a minor reduction of the apparent affinity for Ca(2+). The decrease in the Ca(2+)-ATPase of the mutant enzyme was in parallel with the reduction in the amount of phosphoenzyme formed from Ca(2+) plus ATP. Noteworthy, the mutation nearly eliminated the ability of the enzyme to hydrolyze pNPP which is maximal in the absence of Ca(2+) revealing a major effect of the mutation on the Ca(2+)-independent reactions of the transport cycle. At a pH low enough to protonate the Asp carboxylate the pNPPase activity of Asn879Asp increased, suggesting that the binding of protons to Asn879 is essential for the activities catalyzed by E(2)-like forms of the enzyme.
Rinaldi
Débora E
DE
Instituto de Química y Fisicoquímica Biológicas (IQUIFIB)-Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Ciudad de Buenos Aires, Argentina.
Adamo
Hugo P
HP
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D014638:Vanadates / Q000378:metabolism
D014638:Vanadates; D001216:Asparagine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C507517:ATP2B4 protein, human; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium