Asn792 participates in the hydrogen bond network around the K+-binding pocket of gastric H,K-ATPase

J Biol Chem. 2005 Mar 25;280(12):11488-94. doi: 10.1074/jbc.M412321200. Epub 2005 Jan 11.

Abstract

Asn792 present in M5 of gastric H,K-ATPase is highly conserved within the P-type ATPase family. A direct role in K+ binding was postulated for Na,K-ATPase but was not found in a recent model for gastric H,K-ATPase (Koenderink, J. B., Swarts, H. G. P., Willems, P. H. G. M., Krieger, E., and De Pont, J. J. H. H. M. (2004) J. Biol. Chem. 279, 16417-16424). Therefore, its role in K+ binding and E1/E2 conformational equilibrium in gastric H,K-ATPase was studied by site-directed mutagenesis and expression in Sf9 cells. N792Q and N792A, but not N792D and N792E, had a markedly reduced K+ affinity in both the ATPase and dephosphorylation reactions. In addition, N792A shifted the conformational equilibrium to the E1 form. In double mutants, the effect of N792A on K+ sensitivity was overruled by either E820Q (K(+)-independent activity) or E343D (no dephosphorylation activity). Models were made for the mutants based on the E2 structure of Ca(2+)-ATPase. In the wild-type model the acid amide group of Asn792 has hydrogen bridges to Lys791, Ala339, and Val341. Comparison of the effects of the various mutants suggests that the hydrogen bridge between the carbonyl oxygen of Asn792 and the amino group of Lys791 is essential for the K+ sensitivity and the E2 preference of wild-type enzyme. Moreover, there was a high positive correlation (r = 0.98) between the in silico calculated energy difference of the E2 form (mutants versus wild type) and the experimentally measured IC50 values for vanadate, which reflects the direction of the E2<-->E1 conformational equilibrium. These data strongly support the validity of the model in which Asn792 participates in the hydrogen bond network around the K(+)-binding pocket.

MeSH terms

  • Animals
  • Asparagine
  • Binding Sites
  • Gastric Mucosa / enzymology*
  • H(+)-K(+)-Exchanging ATPase / chemistry*
  • Hydrogen Bonding
  • Imidazoles / pharmacology
  • Models, Molecular
  • Mutation
  • Phosphorylation
  • Potassium / metabolism*
  • Rats
  • Vanadates / pharmacology

Substances

  • Imidazoles
  • Sch 28080
  • Vanadates
  • Asparagine
  • H(+)-K(+)-Exchanging ATPase
  • Potassium