Importance of Na,K-ATPase residue alpha 1-Arg544 in the segment Arg544-Asp567 for high-affinity binding of ATP, ADP, or MgATP.
To identify residues involved in ATP binding in the N-domain of the alpha1-subunit of Na,K-ATPase, mutations were directed to the segment Arg(544)-Asp(567), a beta-strand-loop-helix structure with Arg(544) positioned at the mouth of the ATP-binding pocket near the interface to the P-domain. Substitution of Arg(544) with Gln abolished high-affinity binding of free ATP, while substitution with lysine reduced ADP affinity with minor effects on ATP binding. The contribution of Arg(544) to the change in free energy of ATP binding was estimated to 6.9 kJ/mol (DeltaDeltaG(b)) from double mutations with Asp(369) and to 7.8 kJ/mol from the MgATP dependence of phosphorylation. The phosphorylation data show that binding of Mg(2+) may increase the apparent affinity of wild-type enzyme for ATP [K(1/2)(ATP) 12 nM]. Moderately reduced affinities for ATP were seen after mutations of Asp(555), Glu(556), Asp(565), or Asp(567) with DeltaDeltaG(b) approximately equals 0.5-3 kJ/mol. Mutations of Cys(549) did not affect ATP binding. In conclusion, Arg(544) is important for binding of ATP or ADP, probably by stabilizing the beta- or gamma-phosphate moieties and aligning the gamma-phosphate for interaction with the carboxylate group of Asp(369).
Jacobsen
Mette Dorph
MD
Biomembrane Research Center, Copenhagen University, 2100 Copenhagen OE, Denmark.
Pedersen
Per Amstrup
PA
Jorgensen
Peter Leth
PL
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D008274:Magnesium / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012694:Serine / Q000235:genetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D013816:Thermodynamics
D010446:Peptide Fragments; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D000244:Adenosine Diphosphate; D001216:Asparagine; D000255:Adenosine Triphosphate; D001120:Arginine; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D003545:Cysteine; D000409:Alanine