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

Isaac Wong, Timothy M. Lohman

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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.

Original languageEnglish
Pages (from-to)3115-3125
Number of pages11
Issue number11
StatePublished - Mar 18 1997


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