Abstract
Rapid quench experiments at 25 degrees C were carried out on selected mutants of the sarco(endo)plasmic reticulum Ca(2+)-ATPase to assess the kinetics of the conformational changes of the dephosphoenzyme associated with ATP binding/phosphoryl transfer and the binding and dissociation of Ca(2+) at the cytoplasmically facing transport sites. The mutants Gly(233) --> Glu, Gly(233) --> Val, Pro(312) --> Ala, Leu(319) --> Arg, and Lys(684) --> Arg differed conspicuously with respect to the behavior of the dephosphoenzyme, although they were previously shown to display a common block of the transformation of the phosphoenzyme from an ADP-sensitive to an ADP-insensitive form. The maximum rate of the ATP binding/phosphoryl transfer reaction was reduced 3.6-fold in mutant Gly(233) --> Glu and more than 50-fold in mutant Lys(684) --> Arg, relative to wild type. In mutant Leu(319) --> Arg, the rate of the Ca(2+)-binding transition was reduced as much as 10-30-fold depending on the presence of ATP. In mutants Gly(233) --> Glu, Gly(233) --> Val, and Pro(312) --> Ala, the rate of the Ca(2+)-binding transition was increased at least 2-3-fold at acid pH but not significantly at neutral pH, suggesting a destabilization of the protonated form. The rate of Ca(2+) dissociation was reduced 12-fold in mutant Pro(312) --> Ala and 3.5-fold in Leu(319) --> Arg, and increased at least 4-fold in a mutant in which the putative Ca(2+) liganding residue Glu(309) was replaced by aspartate. The data support a model in which Pro(312) and Leu(319) are closely associated with the cation binding pocket, Gly(233) is part of a long-range signal transmission pathway between the ion-binding sites and the catalytic site, and Lys(684) is an essential catalytic residue that may function in the same way as its counterpart in the soluble hydrolases belonging to the haloacid dehalogenase superfamily.
Highlights
The challenge of understanding active ion transport across biological membranes is well illustrated by the sarco(endo)plasmic reticulum Ca2ϩ-ATPase.1 This enzyme is made up of a single 110-kDa peptide chain that catalyzes uphill Ca2ϩ trans
The residues critical to the E1-P to E2-P conversion are located in various regions of the protein, including the large cytoplasmic loop that makes up the catalytic site, the fourth stalk segment that links the catalytic site to transmembrane segment M4, the -strand domain that interconnects the transmembrane segments M2 and M3, and, to a lesser extent, the transmembrane segments
The aim was to determine the consequences of selected mutations for the kinetics of the conformational changes associated with the ATP binding/phosphoryl transfer reactions and the binding and dissociation of Ca2ϩ at the cytoplasmically facing sites
Summary
Ca2ϩ-ATPase, the sarco(endo)plasmic reticulum Ca2ϩ-transporting adenosine triphosphatase (EC 3.6.1.38); Km, Michaelis constant; M1-M10, putative transmembrane segments numbered from the NH2-terminal end of the peptide chain; MES, 2-(Nmorpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid. We have included in some of the present studies mutant Glu309 Asp, in which the ability to form a stable Ca2ϩ-occluded form in the presence of the ,␥-bidentate chromium(III) complex of ATP was previously found to be lost even though the Ca2ϩactivated phosphorylation by ATP is retained [21]. These mutants display a surprising variability with respect to the characteristics of the reaction steps associated with the dephosphoenzyme
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