The ATP binding site of the yeast plasma membrane proton-translocating ATPase.
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.
Davis
C B
CB
Department of Chemistry, Cornell University, Ithaca, New York 14853.
Smith
K E
KE
Campbell
B N
BN
Hammes
G G
GG
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D005656:Fungal Proteins / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000648:ultrastructure; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010777:Photochemistry; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000345:Affinity Labels; D005656:Fungal Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases