Time-dependent inhibition of recA protein-catalyzed ATP hydrolysis by ATPgammaS: evidence for a rate-determining isomerization of the recA-ssDNA complex.

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