Mutagenesis of segment 487Phe-Ser-Arg-Asp-Arg-Lys492 of sarcoplasmic reticulum Ca2+-ATPase produces pumps defective in ATP binding.
The lysine residue Lys492 located in the large cytoplasmic domain of sarcoplasmic reticulum Ca2+-ATPase is implicated in nucleotide binding through affinity labeling. The contribution of segment 487Phe-Ser-Arg-Asp-Arg-Lys492 to ATP binding and pump function has been investigated through the introduction of 11 site-directed amino acid mutations. ATP binding was measured through competitive inhibition of [gamma-32P]2',3'-O-(2,4, 6-trinitrophenyl)-8-azido-adenosine triphosphate photolabeling of Lys492 or its substitute. Mutations F487S and positional swap F487S/S488F produced pumps that were severely defective in ATP binding (KD > 1 mM), and mutant F487S, together with F487E, exhibited low ATPase activity and low ATP-supported calcium transport and phosphorylation and failed to show CrATP-dependent Ca2+ occlusion. Mutations F487L, R489L, and K492Y were less inhibitory to ATP binding (KD = 8-49 microM) and, together with K492L and R489D/D490R, produced correspondingly smaller changes in ATP-mediated activities. The ATP dependence of ATPase activity of these five mutants showed deviations from the wild-type profile in the low, intermediate, and high concentration ranges, suggesting defects in ATP-dependent conformational changes. Mutations S488A and D490A had no effect on ATP binding (KD = 0.4 microM) or ATP-mediated activities. None of the mutations significantly affected phosphorylation from Pi or acetyl phosphate-supported Ca2+ transport. Mutations F487L and F487S, and not those at residue 492, increased the K0.5 for Ca2+ activation of transport 2- and 8-fold, respectively. The results implicate Phe487, Arg489, and Lys492 in binding ATP in both a catalytic and a regulatory mode.
McIntosh
D B
DB
Department of Chemical Pathology, University of Cape Town Medical School, Cape Town, South Africa.
Woolley
D G
DG
Vilsen
B
B
Andersen
J P
JP
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D019284:Thapsigargin / Q000494:pharmacology
D000345:Affinity Labels; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; C058476:O(2',3')-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium