Phe475 and Glu446 but not Ser445 participate in ATP-binding to the α-subunit of Na+/K+-ATPase

The ATP-binding site of Na+/K+-ATPase is localized on the large cytoplasmic loop of the alpha-subunit between transmembrane helices H-4 and H-5. Site-directed mutagenesis was performed to identify residues involved in ATP binding. On the basis of our recently developed model of this loop, Ser(445), Glu(446), and Phe(475) were proposed to be close to the binding pocket. Replacement of Phe(475) with Trp and Glu(446) with Gln profoundly reduced the binding of ATP, whereas the substitution of Ser(445) with Ala did not affect ATP binding. Fluorescence measurements of the fluorescent analog TNP-ATP, however, indicated that Ser(445) is close to the binding site, although it does not participate in binding. (C) 2002 Elsevier Science (USA). All rights reserved.