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Volume 271, Number 42, Issue of October 18, 1996 pp. 25778-25789
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Mutagenesis of Segment ⁴⁸⁷Phe-Ser-Arg-Asp-Arg-Lys⁴⁹² of Sarcoplasmic Reticulum Ca²⁺-ATPase Produces Pumps Defective in ATP Binding

(Received for publication, May 29, 1996, and in revised form, July 19, 1996)

From the Department of Chemical Pathology, University of Cape Town Medical School, Cape Town, South Africa, and the ^§ Department of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark

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 [-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 (K_D > 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 (K_D = 8-49 µM) 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 (K_D = 0.4 µM) or ATP-mediated activities. None of the mutations significantly affected phosphorylation from P_i or acetyl phosphate-supported Ca²⁺ transport. Mutations F487L and F487S, and not those at residue 492, increased the K_0.5 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.

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