Characterization of the adenosinetriphosphatase activity of the Escherichia coli RecBCD enzyme: relationship of ATP hydrolysis to the unwinding of duplex DNA.
We find that the rate of dsDNA-dependent ATPase activity is biphasic, with a fast component which represents the unwinding of the dsDNA and a slow component which results from the ssDNA-dependent ATPase activity of recBCD enzyme. Comparison of the ATPase and helicase activities permits evaluation of the efficiency of ATP hydrolysis during unwinding. This efficiency can be calculated from the maximum rates of ATPase and helicase activities and is found to range between 2.0 and 3.0 ATP molecules hydrolyzed per base pair of DNA unwound. The number of ATP molecules hydrolyzed per base pair unwound is not altered by temperature but does increase at low concentrations of DNA and high concentrations of sodium chloride and magnesium acetate. The apparent Km values for the DNA and ATP substrates of recBCD enzyme dsDNA-dependent ATPase activity at 25 degrees C were determined to be 0.13 nM DNA molecules and 85 microM ATP, respectively. The observed kcat value is approximately 45 microM ATP s-1 (microM recBCD enzyme)-1. If this rate is corrected for the measured stoichiometry of recBCD enzyme binding to dsDNA, the kcat for ATPase activity corresponds to an ATP hydrolysis rate of approximately 740 ATP molecules s-1 (functional recBCD complex)-1 at 25 degrees C.
Roman
L J
LJ
Department of Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611.
Kowalczykowski
S C
SC
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D013379:Substrate Specificity
D000255:Adenosine Triphosphate; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D000251:Adenosine Triphosphatases; D004264:DNA Topoisomerases, Type I