RecA protein has extremely high cooperativity for substrate in its ATPase activity.
The single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein, especially its cooperativity for ATP, was investigated. To measure the ATPase activity in detail, the methods and reaction conditions for the ATPase assay were reexamined. Under conditions where RecA protein always showed a maximal rate of ATP hydrolysis, its poly(dT)-dependent ATPase activity was measured. At 25 degrees C, increasing the concentration of RecA protein from 0.3 to 1.0 microM increased the turnover number (kcat) from 0.16 to 0.19 s-1 and the Hill coefficient (nH) for ATP from 9.3 to 11.6. At 0.5 microM RecA protein, increasing the temperature from 25 to 37 degrees C increased kcat from 0.18 to 0.35 s-1 but decreased nH from 9.8 to 6.6. Interestingly, the ATPase activity of RecA protein measured in this study showed much higher cooperativity for ATP than those reported to date. Furthermore, the nH value of 11.6 for ATP obtained here was the highest of any ATPase reported so far. These results suggest that the binding of an ATP molecule to a RecA molecule within a nucleoprotein helical filament causes structural change of many other neighboring RecA molecules. This implies that ATP binding induces structural change of the whole nucleoprotein helical filament. Finally, we demonstrated that analysis of cooperativity is useful for revealing how a protein composed of many subunits functions as a whole.
Mikawa
T
T
Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.
Masui
R
R
Kuramitsu
S
S
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D004277:DNA, Single-Stranded; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases