Structural organization and energy transduction mechanism of Na+,K+-ATPase studied with transition metal-catalyzed oxidative cleavage.
This chapter describes contributions of transition metal-catalyzed oxidative cleavage of Na+,K+-ATPase to our understanding of structure-function relations. In the presence of ascorbate/H2O2, specific cleavages are catalyzed by the bound metal and because more than one peptide bond close to the metal can be cleaved, this technique reveals proximity of the different cleavage positions within the native structure. Specific cleavages are catalyzed by Fe2+ bound at the cytoplasmic surface or by complexes of ATP-Fe2+, which directs the Fe2+ to the normal ATP-Mg2+ site. Fe2+- and ATP-Fe2+-catalyzed cleavages reveal large conformation-dependent changes in interactions between cytoplasmic domains, involving conserved cytoplasmic sequences, and a change of ligation of Mg2+ ions between E1P and E2P, which may be crucial in facilitating hydrolysis of E2P. The pattern of domain interactions in E1 and E2 conformations, and role of Mg2+ ions, may be common to all P-type pumps. Specific cleavages can also be catalyzed by Cu2+ ions, bound at the extracellular surfaces, or a hydrophobic Cu2+-diphenyl phenanthroline (DPP) complex, which directs the Cu2+ to the membrane-water interface. Cu2+ or Cu2+-DPP-catalyzed cleavages are providing information on alpha/beta subunit interactions and spatial organization of transmembrane segments. Transition metal-catalyzed cleavage could be widely used to investigate other P-type pumps and membrane proteins and, especially, ATP binding proteins.
Goldshleger
R
R
Department of Biological Chemistry, Weizmann Institute of Science, Rehovoth, Israel.
Patchornik
G
G
Shimon
M B
MB
Tal
D M
DM
Post
R L
RL
Karlish
S J
SJ
D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004735:Energy Transfer / Q000502:physiology*; D008274:Magnesium / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D010084:Oxidation-Reduction; D010758:Phosphorus / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013329:Structure-Activity Relationship
D008670:Metals; D010758:Phosphorus; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium