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 the 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 K-d 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 gelfiltration experiments show that the binding of ADPNP (5'-adenylyl beta,gamma-imidodiphosphate) and coumarins to the 43-kDa protein is mutually 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 similar to 3 x 10(-3) s(-1) and similar to 4 x 10(-3) s(-1) for the 43- and 24-kDa proteins, respectively. Titration calorimetry shows that the K-d values for coumarins binding to both proteins are similar to 10(-8) M and that binding is enthalpy driven.