The Escherichia coli Rep helicase is a dimeric motor protein that catalyzes the transient unwinding of duplex DNA to form single-stranded (ss) DNA using energy derived from the binding and hydrolysis of ATP. In an effort to understand this mechanism of energy transduction, we have used pre-steady-state methods to study the kinetics of ATP binding and hydrolysis by an important intermediate in the DNA unwinding reaction the asymmetric Rep dimer state. P2S, where ss DNA [dT(pT)15] is bound to only one subunit of the Rep dimer. To differentiate between the two potential ATPase active sites inherent in the dimer, we constructed dimers with one subunit covalently cross-linked to ss DNA and where one or the other of the ATPase sites was selectively complexed to the tightly bound transition slate analog ADP-AlF4. We found that when ADP-AlF4 is bound to the Rep subunit in trans from the subunit bound to ss DNA, steady-state ATPase activity of 18 s-1 per dimer (equivalent to wild-type P2S) was recovered. However, when the ADP-AlF4 and ss DNA are both bound to the same subunit (cis), then a titratable burst of ATP hydrolysis is observed corresponding to a single turnover of ATP. Rapid chemical quenched-flow techniques were used to resolve the following minimal mechanism for ATP hydrolysis by the unligated Rep subunit of the cis dimer: E + ATP ⇆ E-ATP ⇆ E'-ATP ⇆ E'-ADP-P(i) ⇆ E-ADP P(i) ⇆ E-ADP + P(i) ⇆ E + ADP + P(i), with K1 = (2.0 ± 0.85) x 105 M-1, k2 = 22 ± 3.5 s-1, k-2 < 0.12 s-1, K3 = 4.0 ± 0.4 (k3 > 200 s-1), k4 = 1.2 ± 0.14 s-1, k-4 << 1.2 s-1, K5 = 1.0 ± 0.2 mM, and K6, = 80 ± 8 μM. A salient feature of this mechanism is the presence of a kinetically trapped long-lived tight nucleotide binding state, E'-ADP-P(i). In the context of our 'subunit switching' model for Rep dimer translocation during processive DNA unwinding [Bjornson, K. B., Wong, I., and Lohman, T. M. (1996) J. Mol. Biol. 263, 411-422], this stale may serve an energy storage function, allowing the energy from the binding and hydrolysis of ATP to be harnessed and held in reserve for DNA unwinding.