title: Differentiation of catalytic sites on Escherichia coli F1ATPase by laser photoactivated labeling with [3H]-2-Azido-ATP using the mutant beta Glu381Cys:epsilonSer108Cys to identify different beta subunits by their interactions with gamma and epsilon subunits.
abstract: The ATP binding affinities of the catalytic sites in the three beta subunits of the Escherichia coli F1 ATPase (ECF1) have been explored in relation to the interaction of these subunits with the small subunits gamma and epsilon. ECF1 from the mutant beta E381C:epsilonS108C was reacted with different concentrations of [3H]-2-azido-ATP and covalent insertion of the nucleotide analogue induced by photoactivation of the azide group to a nitrene with single-pulse UV laser excitation. The enzyme showed cooperative binding of [3H]-2-azido-ATP in the presence of Mg2+. The highest affinity site was located at betafree, the one of the three beta subunits in the mutant that does not form disulfide bonds with either the gamma or the epsilon subunit. This beta subunit is, therefore, the site of unisite catalysis in the enzyme. The second mole of [3H]-2-azido-ATP to bind was located in the beta subunit that links to epsilon (betaepsilon), while the lowest affinity binding of the substrate analogue was with the beta subunit that links to gamma (betagamma). In the absence of Mg2+, all three beta subunits bound [3H]-2-azido-ATP with a similar, low affinity. The results show that binding of MgATP is determined by, and/or must determine, the interactions of the different alpha-beta subunit pairs with the single-copy subunits gamma, delta, and epsilon of the enzyme.
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene, 97403-1229, USA.
authors.forename: G
authors.initials: G
authors.lastname: Grüber
authors.forename: R A
authors.initials: RA
authors.lastname: Capaldi
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels; D001386:Azides / Q000378:metabolism*; D003545:Cysteine; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D018698:Glutamic Acid; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010782:Photolysis; D017354:Point Mutation; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D013050:Spectrometry, Fluorescence; D014316:Tritium
chemical_list: D000345:Affinity Labels; D001386:Azides; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014316:Tritium; D018698:Glutamic Acid; D012694:Serine; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D006180:Proton-Translocating ATPases; D008274:Magnesium; D003545:Cysteine