Energy coupling in DNA gyrase: a thermodynamic limit to the extent of DNA supercoiling.
ATP alpha S (Rp) has been shown to support the supercoiling of plasmid pBR322 catalyzed by Escherichia coli DNA gyrase at comparable rates to the natural substrate ATP and is able to promote the introduction of one more superhelical turn than ATP. The difference in free energy change between consecutive rounds of supercoiling in gyrase-mediated reactions is calculated to be 2.6 kJ mol-1. The difference in free energy of hydrolysis of ATP and ATP alpha S (Rp) has been determined from the difference in the equilibrium constants for the phosphorylation of arginine established by arginine kinase. This equilibrium constant has been found to be displaced by a factor of about 1.5, corresponding to a greater free energy of hydrolysis of ATP alpha S (Rp) compared to ATP of approximately 1 kJ mol-1. This difference in free energy can be tentatively ascribed to a relative destabilization of the MgATP alpha S (Rp) complex with respect to MgATP. Assuming that the stoichiometry of the coupled reactions requires two ATPs hydrolyzed per round of supercoiling, ATP alpha S (Rp) should be capable of providing an additional ca. 2 kJ mol-1 of free energy for DNA supercoiling, which is in good agreement with estimates for the additional free energy required to achieve a further round of supercoiling. These results provide direct evidence to support the proposal that the extent of DNA supercoiling by DNA gyrase is limited by the free energy of hydrolysis of the nucleotide.
Cullis
P M
PM
Department of Chemistry, The University, Leicester, U.K.
Maxwell
A
A
Weiner
D P
DP
D000255:Adenosine Triphosphate / Q000737:chemistry; D001120:Arginine / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004250:DNA Topoisomerases, Type II / Q000737:chemistry*; D004278:DNA, Superhelical / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D010766:Phosphorylation; D010957:Plasmids; D013816:Thermodynamics; D013873:Thionucleotides / Q000737:chemistry
D004278:DNA, Superhelical; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D004250:DNA Topoisomerases, Type II