Gormley N A, Orphanides G, Meyer A, Cullis P M, Maxwell A
Department of Biochemistry, University of Leicester, U.K.
Biochemistry. 1996 Apr 16;35(15):5083-92. doi: 10.1021/bi952888n.
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.
DNA促旋酶是香豆素类抗菌剂的作用靶点。已知这类药物可抑制促旋酶的ATP酶活性，并与促旋酶B蛋白的24 kDa N端亚结构域结合。使用完整DNA促旋酶的超螺旋分析以及使用B蛋白43 kDa N端片段的ATP酶分析表明，这些药物紧密结合，解离常数（Kd）值<10^(-7) M。此外，ATP酶数据表明，1个香豆霉素分子与2个43 kDa蛋白分子相互作用，而其他香豆素则形成1:1复合物。交联实验证实了这一结果。快速凝胶过滤实验表明，ADPNP（5'-腺苷-β,γ-亚氨基二磷酸）和香豆素与43 kDa蛋白的结合是相互排斥的，这与这些药物的竞争作用模式一致。使用24 kDa和43 kDa蛋白进行的快速凝胶过滤结合实验还表明，这些药物的结合缔合速率常数>10^5 M^(-1)·s^(-1)，43 kDa蛋白和解离速率常数约为3×10^(-3) s^(-1)，24 kDa蛋白的解离速率常数约为4×10^(-3) s^(-1)。滴定热分析法表明，香豆素与这两种蛋白结合的Kd值约为十的负八次方摩尔，且结合是由焓驱动的。
