The ATP analog ATPγS is a competitive inhibitor of the recA protein- catalyzed ssDNA-dependent ATP hydrolysis reaction. The degree of inhibition by ATPγS, however, changes in a time-dependent manner and is consistent with a two step binding mechanism. In the first step, ATPγS binds to the recA- ssDNA complex in a rapid equilibrium step (K(D) = 50 μM). This initial binding step is followed by an isomerization of the recA-ssDNA-ATPγS complex to a new conformational state in which ATPγS is bound with a significantly higher affinity (overall K(D) = 0.3 μM). This isomerization is followed by the slow hydrolysis of ATPγS to ADP and thiophosphate (0.01 min-1). The first-order rate constant for the ATPγS-mediated isomerization step (20 min-1), although significantly greater than the rate of ATPγS hydrolysis, is identical to the steady-state rate constant for the recA protein-catalyzed ATP hydrolysis reaction. These results are consistent with a kinetic model in which an ATP-mediated isomerization of the recA-ssDNA complex represents the rate-determining step on the recA protein-catalyzed ssDNA-dependent ATP hydrolysis reaction pathway.
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