Bicyclomycin and dihydrobicyclomycin inhibition kinetics of Escherichia coli rho-dependent transcription termination factor ATPase activity.
Bicyclomycin and dihydrobicyclomycin inhibition kinetics of Escherichia coli rho-dependent transcription termination factor ATPase activity.
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双环霉素和二氢双环霉素对大肠杆菌 rho 依赖性转录终止因子 ATPase 活性的抑制动力学。
DOI：
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发表时间：
1995
影响因子：
3.9
通讯作者：
H. Kohn
中科院分区：
文献类型：
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作者：
H. G. Park;X. Zhang;H. Moon;A. Zwiefka;K. Cox;S. Gaskell;W. Widger;H. Kohn
新型抗生素双环霉素在大肠杆菌中的主要作用位点已被确定为 rho 转录终止因子。已发现双环霉素对 rho poly(C) 刺激的 ATP 水解的抑制是通过相对于 ATP 的非竞争性、可逆途径进行的 (Ki = 20 microM)。二氢双环霉素的抑制作用相似（Ki = 75 microM）。在两种 rho 突变体 Cys202Gly 和 Cys202Ser 的测定条件下，未观察到抗生素的抑制特性发生变化，表明 Cys-202 不影响药物与 rho 的结合。野生型 rho 与双环霉素 (20 mM) 长时间孵育（32 摄氏度，12 小时）会导致蛋白质降解，并且透析后 rho ATP 酶活性缓慢、永久丧失。获得的证据表明双环霉素中存在的痕量蛋白酶是观察到的蛋白质降解的原因。用纯化的双环霉素 (25 mM) 处理野生型和突变型 rho 蛋白，导致透析后 ATP 酶活性损失约 80%，但蛋白质没有明显损失。然而，与野生型 rho 相比，双环霉素处理的 rho 的电泳迁移率有所降低。在野生型 rho 中添加 ATP 或 Poly(C) 可部分防止双环霉素失活，而包含两种配体可提供近乎完全的防止双环霉素失活的保护。 rho 与过量纯化双环霉素长时间孵育后观察到的 ATP 酶活性损失归因于抗生素在多个位点对蛋白质的共价修饰。
The primary site of action for the novel antibiotic, bicyclomycin, in Escherichia coli has been identified to be the rho transcription termination factor. The inhibition of rho poly(C)-stimulated hydrolysis of ATP by bicyclomycin has been found to proceed by a non-competitive, reversible pathway with respect to ATP (Ki = 20 microM). Inhibition by dihydrobicyclomycin was similar (Ki = 75 microM). No change in the inhibitory properties of the antibiotic was observed under the assay conditions with the two rho mutants, Cys202Gly and Cys202Ser, indicating that Cys-202 does not affect drug binding to rho. Prolonged incubation (32 degrees C, 12 h) of wild-type rho with bicyclomycin (20 mM) led to protein degradation and a slow, permanent loss of rho ATPase activity after dialysis. Evidence was obtained that trace amounts of proteases present with bicyclomycin were responsible for the observed protein degradation. Treatment of wild-type and mutant rho proteins with purified bicyclomycin (25 mM) led to approximately 80% loss of ATPase activity after dialysis with no apparent loss of protein. However, a reduction of the electrophoretic mobility of the bicyclomycin-treated rho versus wild-type rho was seen. Addition of either ATP or poly(C) to wild-type rho led to partial protection against bicyclomycin inactivation, while inclusion of both ligands provided near complete protection against inactivation. The observed loss of ATPase activity upon prolonged incubation of rho with excess purified bicyclomycin is attributed to the covalent modification of the protein by the antibiotic at multiple sites.