Interaction of nucleotides with Asp(351) and the conserved phosphorylationloop of sarcoplasmic reticulum Ca2+-ATPase

The nucleotide binding properties of mutants with alterations to Asp(351) a nd four of the other residues in the conserved phosphorylation loop, (351)D KTGTLT(357), Of sarcoplasmic reticulum Ca2+-ATPase were investigated using an assay based on the 2',3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triph osphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP, In selected cases where the competition assay showed extremely high affini ty, ATP binding was also measured by a direct filtration assay, At pH 8.5 i n the absence of Ca2+,mutations removing the negative charge of Asp351 (D35 1N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP w ith affinities 20-156-fold higher than wild type (K-D as low as 0.006 mu M) , whereas the affinity of mutant D351E was comparable with wild type. Mutat ions K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTN P-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T complete ly prevented photolabeling of Lys492. In the absence of Ca2+, mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of M g2+ and at alkaline pH (El state). The affinity of mutant D351A for MgATP w as extraordinarily high in the presence of Ca2+ (K-D = 0.001 mu M), suggest ing a transition state like configuration at the active site under these co nditions. The mutants with reduced ATP affinity, as well as mutants D351N a nd D351A, exhibited reduced or zero CrATP-induced Ca2+ occlusion due to def ective CrATP binding.