The phosphatase activity of the isolated H4‐H5 loop of Na+/K+ ATPase resides outside its ATP binding site

Abstract

The structural stability of the large cytoplasmic domain (H4-H5 loop) of mouse α1 subunit of Na+/K+ ATPase (L354–I777), the number and the location of its binding sites for 2′-3′-O-(trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) and p-nitrophenylphosphate (pNPP) were investigated. C- and N-terminal shortening revealed that neither part of the phosphorylation (P)-domain are necessary for TNP-ATP binding. There is no indication of a second ATP site on the P-domain of the isolated loop, even though others reported previously of its existence by TNP-N3ADP affinity labeling of the full enzyme. Fluorescein isothiocyanate (FITC)-anisotropy measurements reveal a considerable stability of the nucleotide (N)-domain suggesting that it may not undergo a substantial conformational change upon ATP binding. The FITC modified loop showed only slightly diminished phosphatase activity, most likely due to a pNPP site on the N-domain around N398 whose mutation to D reduced the phosphatase activity by 50%. The amino acids forming this pNPP site (M384, L414, W411, S400, S408) are conserved in the α1−4 isoforms of Na+/K+ ATPase, whereas N398 is only conserved in the vertebrates' α1 subunit. The phosphatase activity of the isolated H4-H5 loop was neither inhibited by ATP, nor affected by mutation of D369, which is phosphorylated in native Na+/K+ ATPase.