Energy coupling in DNA gyrase and the mechanism of action of novobiocin.
Energy coupling in DNA gyrase and the mechanism of action of novobiocin.
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DNA 旋转酶中的能量耦合和新生霉素的作用机制。
DOI：
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发表时间：
1978
影响因子：
11.1
通讯作者：
N. Cozzarelli
中科院分区：
文献类型：
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作者：
A. Sugino;Higgins Np;P. Brown;Peebles Cl;N. Cozzarelli
大肠杆菌DNA旋转酶以ATP为代价催化闭合双链DNA的负超螺旋。阐明超螺旋反应的能量偶联组分的酶的另外两种活性是ATP到ADP和P（i）的DNA依赖性水解以及由ATP改变促旋酶切割反应的DNA位点特异性。这两条DNA链的切割是由十二烷基硫酸钠处理被抑制剂oxolinic酸捕获的稳定的回旋酶-DNA复合物引起的。ATP或不可水解的类似物Adenyl-5 '-yl-imidodiophosphate（App[NH]p）移动ColE 1 DNA上的主要切割位点。新生霉素和香豆霉素A（1）阻止了这种裂解重排，使抗生素的作用位点位于ATP水解之前的反应步骤。阻断的步骤是ATP的结合，因为在ATP酶和超螺旋测定中，香豆霉素A（1）和新生霉素与ATP竞争性相互作用; K（i）值比ATP的K（m）小四个数量级以上。这个简单的机制解释了药物对DNA促旋酶的所有影响。对另一种由促旋酶催化的反应的有效竞争性抑制剂App[NH]p的研究表明，驱动DNA进入更高能量的超螺旋形式并不需要切割高能键。在促旋酶的底物水平下，App[NH]p诱导与酶量成比例的超螺旋;每个促旋酶原聚体A引入-0.3超螺旋转角。我们假设ATP和App[NH]p是导致一轮超螺旋的促旋酶构象变化的变构效应物。ATP水解促进的核苷酸解离使促旋酶返回其原始构象，从而允许酶周转。这种伴随核苷酸亲和力改变的环状构象变化似乎也是其他不同过程（包括肌肉收缩、蛋白质合成和氧化磷酸化）中能量转导的共同特征。
Escherichia coli DNA gyrase catalyzes negative supercoiling of closed duplex DNA at the expense of ATP. Two additional activities of the enzyme that have illuminated the energy coupling component of the supercoiling reaction are the DNA-dependent hydrolysis of ATP to ADP and P(i) and the alteration by ATP of the DNA site specificity of the gyrase cleavage reaction. This cleavage of both DNA strands results from treatment with sodium dodecyl sulfate of the stable gyrase-DNA complex that is trapped by the inhibitor oxolinic acid. Either ATP or a nonhydrolyzable analogue, adenyl-5'-yl-imidodiphosphate (App[NH]p), shifts the primary cleavage site on ColE1 DNA. The prevention by novobiocin and coumermycin A(1) of this cleavage rearrangement places the site of action of the antibiotics at a reaction step prior to ATP hydrolysis. The step blocked is the binding of ATP because coumermycin A(1) and novobiocin interact competitively with ATP in the ATPase and supercoiling assays; the K(i) values are more than four orders of magnitude less than the K(m) for ATP. This simple mechanism accounts for all effects of the drugs on DNA gyrase. Studies with App[NH]p, another potent competitive inhibitor of reactions catalyzed by gyrase, show that cleavage of a high energy bond is not required for driving DNA into the higher energy supercoiled form. With substrate levels of gyrase, App[NH]p induces supercoiling that is proportional to the amount of enzyme; a -0.3 superhelical turn was introduced per gyrase protomer A. We postulate that ATP and App[NH]p are allosteric effectors of a conformational change of gyrase that leads to one round of supercoiling. Nucleotide dissociation favored by hydrolysis of ATP returns gyrase to its original conformation and thereby permits enzyme turnover. Such cyclic conformational changes accompanying alteration in nucleotide affinity also seem to be a common feature of energy transduction in other diverse processes including muscle contraction, protein synthesis, and oxidative phosphorylation.