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RecA protein has extremely high cooperativity for substrate in its ATPase activity
T Mikawa1, R Masui, S Kuramitsu
1Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.
Journal of Biochemistry
|May 30, 1998
Summary
Investigating the ATPase activity of Escherichia coli RecA protein revealed exceptionally high cooperativity for ATP. This suggests ATP binding induces significant structural changes throughout the entire RecA nucleoprotein filament.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- The RecA protein from Escherichia coli is crucial for DNA repair and recombination.
- Understanding its ATPase activity and cooperativity is key to elucidating its functional mechanisms.
Purpose of the Study:
- To investigate the single-stranded DNA-dependent ATPase activity and ATP cooperativity of E. coli RecA protein.
- To reexamine assay methods and conditions for detailed ATPase activity measurement.
Main Methods:
- Reexamination of ATPase assay methods and reaction conditions.
- Measurement of poly(dT)-dependent ATPase activity of RecA protein under maximal rate conditions.
- Analysis of turnover number (kcat) and Hill coefficient (nH) for ATP at varying RecA concentrations and temperatures.
Main Results:
- Increased RecA protein concentration enhanced turnover number (kcat) and ATP cooperativity (nH).
- Elevated temperature increased kcat but decreased nH.
- Observed ATP cooperativity (nH up to 11.6) was significantly higher than previously reported for any ATPase.
Conclusions:
- The high cooperativity suggests ATP binding induces substantial structural changes in the RecA nucleoprotein helical filament.
- Analysis of cooperativity provides insights into the functional mechanisms of multi-subunit proteins like RecA.