The rate-limiting transition state of steady-state ATP hydrolysis and synthesis reactions in the F(o)F(1) ATP synthase involves the rotation of the gamma, epsilon, and c subunits. To probe the role of the transport and coupling mechanisms in controlling catalysis, kinetic and thermodynamic parameters of ATP hydrolysis were determined for enzymes in the presence of the detergent lauryldimethylamine oxide (LDAO), which uncouples active transport and disables the inhibitory effect of the epsilon subunit. At 5 mM LDAO or greater, the inhibitory effects of epsilon subunit are abrogated in both purified F(1) and membranous F(o)F(1). In these conditions, LDAO solubilized F(o)F(1) has a higher k(cat) for ATP hydrolysis than F(1). These results indicate an influence of F(o) on F(1) even though catalysis is uncoupled from transport. The alpha(3)beta(3)gamma complex free of the epsilon subunit is activated at a lower concentration of 0.5 mM LDAO. Significantly, the gammaY205C mutant enzyme is similarly activated at 0.5 mM LDAO, suggesting that the mutant enzyme lacks epsilon inhibition. The gammaY205C F(o)F(1), which has a k(cat) for ATP hydrolysis 2-fold higher than wild type, has an ATP synthesis rate 3-fold lower than wild type, showing that coupling is inefficient. Arrhenius and isokinetic analyses indicate that enzymes that are free of epsilon subunit inhibition have a different transition-state structure from those under the influence of the epsilon subunit. We propose that the epsilon subunit is one of the factors that determines the proper transition-state structure, which is essential for efficient coupling.