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-SUBUNIT AND EPSILON-SUBUNIT

The ATP binding affinities of the catalytic sites in the three beta su bunits of the Escherichia coli F-1 ATPase (ECF(1)) have been explored in relation to the interaction of these subunits with the small subuni ts gamma and epsilon. ECF(1) from the mutant beta E381C:epsilon S108C was reacted with different concentrations of [H-3]-2-azido-ATP and cov alent 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 pre sence 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 di sulfide bonds with either the gamma or the epsilon subunit. This beta subunit is, therefore, the site of unisite catalysis in the enzyme. Th e second mole of [H-3]-2-azido-ATP to bind was located in the beta sub unit 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 subuni ts bound [H-3]-2-azido-ATP with a similar, low affinity. The results s how that binding of MgATP is determined by, and/or must determine, the interactions of the different alpha-beta subunit pairs with the singl e-copy subunits,gamma, delta and epsilon of the enzyme.