The interaction of coumarin antibiotics with fragments of DNA gyrase B protein.
DNA gyrase is the target of the coumarin group of antibacterial agents. The drugs are known to inhibit the ATPase activity of gyrase and bind to the 24-kDa N-terminal subdomain of gyrase B protein. Supercoiling assays with intact DNA gyrase and ATPase assays with a 43-kDa N-terminal fragment of the B protein suggest that the drugs bind tightly, with Kd values <10(-7) M. In addition, the ATPase data suggest that 1 coumermycin molecule interacts with 2 molecules of the 43-kDa protein while the other coumarins form a 1:1 complex. This result is confirmed by cross-linking experiments. Rapid gel-filtration experiments show that the binding of ADPNP(5'-adneylyl beta,gamm-imidodiphosphate) and coumarins to the 43-kDa protein is mutally exclusive, consistent with a competitive mode of action for the drugs. Rapid gel-filtration binding experiments using both the 24-and 43-kDa proteins also show that the drugs bind with association rate constants of >10(5) M-1.s-1, and dissociation rate constants of approximately 3x10(-3)s-1 and approximately 4x10(-3)s-1 for the 43-and 24-kDa proteins, respectively. Titration calorimetry shows that the Kd values for coumarins binding to both proteins are approximately 10-8M and that binding is enthalpy driven.
Gormley
N A
NA
Department of Biochemistry, University of Leicester, U.K.
Orphanides
G
G
Meyer
A
A
Cullis
P M
PM
Maxwell
A
A
D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D049933:Aminocoumarins; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D003374:Coumarins / Q000378:metabolism / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000378:metabolism / Q000494:pharmacology*; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D007700:Kinetics; D009675:Novobiocin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D017510:Protein Folding; D059005:Topoisomerase II Inhibitors
D049933:Aminocoumarins; D000900:Anti-Bacterial Agents; D003374:Coumarins; D003432:Cross-Linking Reagents; D059005:Topoisomerase II Inhibitors; D009675:Novobiocin; D000266:Adenylyl Imidodiphosphate; C099094:dihydronovobiocin; C006260:clorobiocin; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; C004628:coumermycin