title: 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 (H(4)-H(5) loop) of mouse alpha(1) 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-N(3)ADP 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 alpha(1-4) isoforms of Na(+)/K(+) ATPase, whereas N398 is only conserved in the vertebrates' alpha(1) subunit. The phosphatase activity of the isolated H(4)-H(5) loop was neither inhibited by ATP, nor affected by mutation of D369, which is phosphorylated in native Na(+)/K(+) ATPase.
authors.affiliation: Institute of Biochemistry and Endocrinology, Justus-Liebig-University Giessen, D-35392 Giessen, Germany.
authors.forename: Rita
authors.initials: R
authors.lastname: Krumscheid
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Ettrich
authors.forename: Zofie
authors.initials: Z
authors.lastname: Sovová
authors.forename: Klára
authors.initials: K
authors.lastname: Susánková
authors.forename: Zdenek
authors.initials: Z
authors.lastname: Lánský
authors.forename: Katerina
authors.initials: K
authors.lastname: Hofbauerová
authors.forename: Holger
authors.initials: H
authors.lastname: Linnertz
authors.forename: Jan
authors.initials: J
authors.lastname: Teisinger
authors.forename: Evzen
authors.initials: E
authors.lastname: Amler
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schoner
mesh_terms: D009597:4-Nitrophenylphosphatase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003593:Cytoplasm / Q000201:enzymology; D004795:Enzyme Stability; D004801:Eosine Yellowish-(YS) / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine
chemical_list: D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D009597:4-Nitrophenylphosphatase; D017027:Protein Tyrosine Phosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D004801:Eosine Yellowish-(YS)