title: Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase.
abstract: The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding.
authors.affiliation: Department of Chemical Pathology, University of Cape Town Medical School, 7925 Cape Town, South Africa. davidmci@chempath.uct.ac.za
authors.forename: D B
authors.initials: DB
authors.lastname: McIntosh
authors.forename: D G
authors.initials: DG
authors.lastname: Woolley
authors.forename: D H
authors.initials: DH
authors.lastname: MacLennan
authors.forename: B
authors.initials: B
authors.lastname: Vilsen
authors.forename: J P
authors.initials: JP
authors.lastname: Andersen
mesh_terms: D000818:Animals; D001218:Aspartame; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
chemical_list: D009711:Nucleotides; D000252:Calcium-Transporting ATPases; D001218:Aspartame