Differentiation of catalytic sites on Escherichia coli F₁ATPase by laser photoactivated labeling with [³H]-2-azido-ATP using the mutant βGlu381Cys:εSer108Cys to identify different β subunits by their interactions with γ and ε subunits

The ATP binding affinities of the catalytic sites in the three β subunits of the Escherichia coli F₁ ATPase (ECF₁) have been explored in relation to the interaction of these subunits with the small subunits γ and ε. ECF₁ from the mutant βE381C:εS108C 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 β_free, the one of the three β subunits in the mutant that does not form disulfide bonds with either the γ or the ε subunit. This β 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 β subunit that links to ε (β_ε), while the lowest affinity binding of the substrate analogue was with the β subunit that links to γ (β_γ). In the absence of Mg²⁺, all three β 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 α-β subunit pairs with the single-copy subunits, γ, δ, and ε of the enzyme.