The rate-determining step on the recA protein-catalyzed ssDNA-dependent ATP hydrolysis reaction pathway.
We recently constructed a mutant recA protein in which His 163 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 ATPgammaS-mediated conformational transitions of the [H163W]recA-ssDNA complex. In this report, the pre-steady-state kinetics of the ATP and ATPgammaS-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 is identical to the steady-state turnover number for ATP hydrolysis. The kinetics of the ATPgammaS-mediated fluorescence change were also consistent with a two-step binding mechanism with a unimolecular isomerization of 18 min(-1); since ATPgammaS is not hydrolyzed appreciably on the time scale of these experiments (0.017 min(-1)), this indicates that the isomerization step follows ATPgammaS (or ATP) binding but precedes ATPgammaS (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.
Stole
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Department of Biochemistry, The Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205, USA.
Bryant
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FR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D006639:Histidine; D006868:Hydrolysis; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011938:Rec A Recombinases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D006639:Histidine; D014364:Tryptophan; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases