Open AccessThe complex ATP–Fe 2+ serves as a specific affinity cleavage reagent in ATP-Mg 2+ sites of Na,K-ATPase: Altered ligation of Fe 2+ (Mg 2+ ) ions accompanies the E 1 P→E 2 P conformational change
Author(s) -
Guy Patchornik,
Rivka Goldshleger,
Steven J. D. Karlish
Publication year - 2000
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.220332897
Subject(s) - chemistry , cleavage (geology) , crystallography , stereochemistry , nucleotide , atp hydrolysis , atpase , reagent , enzyme , biochemistry , biology , paleontology , fracture (geology) , gene
In the presence of ascorbate/H2 O2 , ATP–Fe2+ or AMP-PNP–Fe2+ complexes act as affinity cleavage reagents, mediating selective cleavage of the alpha subunit of Na,K-ATPase at high affinity ATP–Mg2+ sites. The cleavages reveal contact points of Fe2+ or Mg2+ ions. In E1 and E1 Na conformations, two major cleavages are detected within the conserved708 TGDGVNDSPALKK sequence (at V712 and nearby), and one (E1 Na) or two (E1 ) minor cleavages near V440. In media containing sodium and ATP, Fe2+ substitutes for Mg2+ in activating phosphorylation and ATP hydrolysis. In the E1 P conformation, cleavages are the same as in E1 . Fe2+ is not bound tightly. By contrast, in the E2 P conformation, the pattern is different. A major cleavage occurs near the conserved sequence212 TGES, whereas those in TGDGVNDSPALKK are less prominent. Fe2+ is bound very tightly. On E2 P hydrolysis, the Fe2+ dissociates. The results are consistent with E1 ↔E2 conformation-dependent movements of cytoplasmic domains and sites for Pi and Mg2+ ions, inferred from previous Fe-cleavage experiments. Furthermore, these concepts fit well with the crystal structure of Ca-ATPase [Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000)Nature (London) 405, 647–655]. Altered ligation of Mg2+ ions in E2 P may be crucial in facilitating nucleophilic attack of water on the O—P bond. Mg2+ ions may play a similar role in all P-type pumps. As affinity cleavage reagents, ATP–Fe2+ or other nucleotide–Fe2+ complexes could be widely used to investigate nucleotide binding proteins.