A two-site mechanism for ATP hydrolysis by the asymmetric Rep dimer P2S as revealed by site-specific inhibition with ADP-AlF4

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 state 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 reversible arrow E-ATP reversible arrow E'-ATP reversible arrow E'-ADP-P-i reversible arrow E-ADP-P-i reversible arrow E-ADP+P-i reversible arrow E+ADP+P-i, with K-1 = (2.0 +/- 0.85) x 10(5) M(-1), k(2) = 22 +/- 3.5 s(-1), k(-2) < 0.12 s(-1), K-3 = 4.0 +/- 0.4 (k(3) > 200 s(-1)), k(4) = 1.2 +/- 0.14 s(-1), k(-4) much less than 1.2 s(-1), K-5 = 1.0 +/- 0.2 mM, and K-6 = 80 +/- 8 mu M. 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., & Lohman, T. M. (1996) J. Mol. Biol. 263, 411-422], this state 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.