Yoshida H, Nakamura M, Bogaki M, Ito H, Kojima T, Hattori H, Nakamura S
Bioscience Research Laboratories, Dainippon Pharmaceutical Co., Ltd., Osaka, Japan.
Antimicrob Agents Chemother. 1993 Apr;37(4):839-45. doi: 10.1128/AAC.37.4.839.
The mechanism of action of quinolones was investigated by use of various DNA gyrases reconstituted from wild-type and mutant GyrA and GyrB proteins of Escherichia coli. The quinolone sensitivities of the DNA supercoiling activity of the gyrases were generally parallel to the quinolone susceptibilities of strains having the corresponding enzymes and depended on gyrase subunits but not on substrate DNA. [3H]Enoxacin did not bind to gyrase alone or DNA alone but bound to gyrase-DNA complexes when measured by a gel filtration method. There appeared to be two enoxacin binding phases, at low and high enoxacin concentrations, for the wild-type gyrase-DNA and type 2 GyrB (Lys-447 to Glu) mutant gyrase-DNA complexes but only one enoxacin binding phase at the concentrations used for the GyrA (Ser-83 to Leu) mutant gyrase-DNA and type 1 GyrB (Asp-426 to Asn) mutant gyrase-DNA complexes. New enoxacin binding sites appeared in the presence of enoxacin, and the enoxacin binding affinities for the sites, especially at low enoxacin concentrations, near the MICs for the strains having the corresponding gyrases, correlated well with the enoxacin sensitivities of the gyrases and the MICs. From the results obtained, we propose a quinolone pocket model as the mechanism of action of quinolones, in which quinolones exert their action through binding to a gyrase-DNA complex and the quinolone binding affinities for the complex are determined by both GyrA and GyrB subunits in concert.
利用从大肠杆菌野生型和突变型GyrA及GyrB蛋白重构的各种DNA促旋酶，对喹诺酮类药物的作用机制进行了研究。促旋酶的DNA超螺旋活性对喹诺酮类药物的敏感性通常与具有相应酶的菌株对喹诺酮类药物的敏感性平行，并且取决于促旋酶亚基，而不取决于底物DNA。通过凝胶过滤法测定，[3H]依诺沙星不单独与促旋酶或DNA结合，而是与促旋酶-DNA复合物结合。对于野生型促旋酶-DNA和2型GyrB（赖氨酸-447突变为谷氨酸）突变型促旋酶-DNA复合物，在低和高依诺沙星浓度下似乎存在两个依诺沙星结合阶段，但对于GyrA（丝氨酸-83突变为亮氨酸）突变型促旋酶-DNA和1型GyrB（天冬氨酸-426突变为天冬酰胺）突变型促旋酶-DNA复合物，在所用浓度下仅存在一个依诺沙星结合阶段。在依诺沙星存在的情况下出现了新的依诺沙星结合位点，并且这些位点对依诺沙星的结合亲和力，尤其是在低依诺沙星浓度下，接近具有相应促旋酶的菌株的最低抑菌浓度（MIC），与促旋酶对依诺沙星的敏感性和MIC密切相关。根据所得结果，我们提出了一种喹诺酮口袋模型作为喹诺酮类药物的作用机制，其中喹诺酮类药物通过与促旋酶-DNA复合物结合发挥作用，并且喹诺酮对该复合物的结合亲和力由GyrA和GyrB亚基共同决定。
