Differentiation of catalytic sites on Escherichia coli F(1)ATPase by laser photoactivated labeling with [H-3]-2-azido-ATP using the mutant beta Glu381Cys:epsilon Ser108Cys to identify different beta subunits by their interactions with gamma and epsilon subunits

The ATP binding affinities of the catalytic sites in the three beta subunits of the Escherichia coli F-1 ATPase (ECF(1)) have been explored in relation to the interaction of these subunits with the small subunits gamma and epsilon. ECF(1) from the mutant beta E381C:epsilon S108C was reacted with different concentrations of [H-3]-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 [H-3]-2-azido-ATP in the presence of Mg2(+). The highest affinity site was located at beta(free), 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 [H-3]-2-azido-ATP to bind was located in the beta subunit that links to epsilon (beta(epsilon)), while the lowest affinity binding of the substrate analogue was with the beta subunit that links to gamma (beta(gamma)). In the absence of Mg2+, all three beta subunits bound [H-3]-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.