title: Functional role of oxygen-containing residues in the fifth transmembrane segment of the Na,K-ATPase alpha subunit.
abstract: The functional roles of Tyr771, Thr772, and Asn776 in the fifth transmembrane segment of the Na, K-ATPase alpha subunit were studied using site-directed mutagenesis, expression, and kinetics analysis. Nonconservative replacements Thr772Tyr and Asn776Ala led to reduced Na,K-ATPase turnover. Replacements at these positions (Asn776Ala, Thr772Leu, and Thr772Tyr) also led to high Na-ATPase activity (in the absence of K+). However, Thr772- and Asn776-substituted enzymes showed only small alterations in the apparent Na+ and K+ affinities (K1/2 for Na,K-ATPase activation). Thus, the high Na-ATPase activity does not appear related to cation-binding alterations. It is probably associated with conformational alterations which lead to an acceleration of enzyme dephosphorylation by Na+ acting at the extracellular space (Argüello et al. J. Biol. Chem. 271, 24610-24616, 1996). Nonconservative substitutions at position 771 (Tyr771Ala and Tyr771Ser) produced a significant decrease of enzyme turnover. Enzyme-Na+ interaction was greatly changed in these enzymes, while their activation by K+ did not appear affected. Although the Na+ K1/2 for Na,K-ATPase stimulation was unchanged (Tyr771Ala, Tyr771Ser), the activation by this cation showed no cooperativity (Tyr771Ala, nHill = 0.75; Tyr771Ser, nHill = 0.92; Control, nHill = 2.28). Substitution Tyr771Phe did not lead to a significant reduction in the cooperativity of the ATPase Na+ dependence (nHill = 1.91). All Tyr771-substituted enzymes showed low steady-state levels of phosphoenzyme during Na-activated phosphorylation by ATP. Phosphorylation levels were not increased by oligomycin, although the drug bound and inactivated Tyr771-substituted enzymes. No E1 left and right arrow E2 equilibrium alterations were detected using inhibition by vanadate as a probe. The data suggest that Tyr771 might play a central role in Na+ binding and occlusion without participating in K+-enzyme interactions.
authors: Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts, 01609, USA. arguello@wpi.edu
authors: J M
authors: JM
authors: Argüello
authors: J
authors: J
authors: Whitis
authors: M C
authors: MC
authors: Cheung
authors: J B
authors: JB
authors: Lingrel
mesh_terms: D019943:Amino Acid Substitution / Q000502:physiology*; D001216:Asparagine / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000502:physiology*; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000502:physiology*; D013912:Threonine / Q000502:physiology; D014443:Tyrosine / Q000502:physiology
chemical_list: D013912:Threonine; D014443:Tyrosine; D001216:Asparagine; D000254:Sodium-Potassium-Exchanging ATPase; D010100:Oxygen