We recently constructed a mutant recA protein in which His163 was replaced by a tryptophan residue. The [H163W]recA protein is functionally identical to the wild-type protein, and the Trp163 side chain serves as a fluorescence reporter group for the ATP and ATPγS-mediated conformational transitions of the [H163W]recA-ssDNA complex. In this report, the pre- steady-state kinetics of the ATP and ATPγS-mediated transitions were examined by stopped-flow fluorescence. The kinetics of the ATP-mediated fluorescence change were consistent with a two-step mechanism in which an initial rapid equilibrium binding of ATP to the recA-ssDNA complex is followed by a first-order isomerization of the complex to a new conformational state; the rate constant for the isomerization step of 18 min-1 is identical to the steady-state turnover number for ATP hydrolysis. The kinetics of the ATPγS-mediated fluorescence change were also consistent with a two-step binding mechanism with a unimolecular isomerization of 18 min-1; since ATPγS is not hydrolyzed appreciably on the time scale of these experiments (0.017 min-1), this indicates that the isomerization step follows ATPγS (or ATP) binding but precedes ATPγS (or ATP) hydrolysis. These and other results are consistent with a kinetic model in which an ATP- mediated isomerization of the recA-ssDNA complex is the rate-determining step on the recA protein-catalyzed ssDNA-dependent ATP hydrolysis reaction pathway.
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