The lysine residue Lys(492) located in the large cytoplasmic domain of sarcoplasmic reticulum Ca2+-ATPase is implicated in nucleotide binding through affinity labeling, The contribution of segment (487)Phe-Ser Arg-Asp-Arg Lys(492) 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-P-32]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine triphosphate photolabeling of Lys(492) 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 mu M) 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 mu M) 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 Phe(487), Arg(489) and Lys(492) in binding ATP in both a catalytic and a regulatory mode.
