Photoaffinity labeling of the active site of the yeast plasma membrane H(+)-ATPase has been studied with 2-azido-AMP and 2-azido-ATP. The ATPase activity of the enzyme decreases as the time of photolysis of the photoactive nucleotides in the presence of the enzyme increases. The covalent incorporation of [alpha-32P]2-azido-AMP into the enzyme and the inhibition of ATPase activity have comparable time courses. ATP protects the ATPase from incorporation of and photoinactivation by 2-azido-ATP or 2-azido-AMP. In the dark, 2-azido-ATP inhibits the ATPase at concentrations comparable to the apparent Michaelis constant for MgATP. After photolysis and proteolysis of the protein, three overlapping peptides labeled by the nucleotide analogues were purified by reversed-phase high performance liquid chromatography and sequenced. The peptides are derived from a region of the ATPase that is highly conserved in related cation pumps forming a phosphorylated intermediate during the catalytic cycle. Labeling with both nucleotide analogues occurs in peptides containing residues from aspartate 560 to lysine 566. The amino acids in this region conform to a consensus sequence for ATP binding derived from phosphofructokinase.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|