Department of Chemistry, National Chung Hsing University, Taichung, Taiwan 40227, Rep. of China.
Chemistry. 2011 Feb 1;17(5):1493-506. doi: 10.1002/chem.201002330. Epub 2010 Dec 16.
The antibiotic neocarzinostatin comprises a carrier protein with a well-defined cavity for accommodating an active enediyne chromophore. The protein has two disulfides, one (Cys(37)-Cys(47)) lies on the cavity bottom and the other (Cys(88)-Cys(93)) in a constrained short loop. When the chromophore is not bound to the protein, a thiol-induced cycloaromatization of the enediyne into a tetrahydroindacene derivative is responsible for the potent antitumor activity. When it is protein-bound, the protein diverts the cycloaromatization pathway to form a distinct hydroxyisochromene-type product. How the protein directs the enediyne chemistry is an interesting puzzle, and various suggestions have been proposed in the past. We screened more than fifty thiols and manipulated conditions to locate reaction features and search for factors that could influence the protein directing strength. Thiol- and oxygen-concentration-dependence studies suggested that disulfides, which maintain the steric rigidity of the protein, could play a key role in diverting the cycloaromatization pathway. For direct proofs, we made mutations at each of the two disulfides by replacing sulfur atoms with oxygen. Circular dichroism and two-dimensional NMR spectroscopy studies suggested that the mutations changed neither the protein conformation nor the ligand interactions. Analyses of the thiol-induced cycloaromatization revealed that rupture of Cys(37)-Cys(47) made the protein almost completely lose its chemical directing ability, whereas rupture of Cys(88)-Cys(93) had only a minor influence. The results demonstrated that the steric rigidity of the binding cavity, but not necessary the whole protein, played an important role in the protein-directed mechanism.
抗生素新制癌菌素由一个载体蛋白组成，该蛋白具有一个明确的腔，用于容纳活性烯二炔类发色团。该蛋白有两个二硫键，一个（Cys(37)-Cys(47)）位于腔底部，另一个（Cys(88)-Cys(93)）位于受约束的短环中。当发色团未与蛋白结合时，烯二炔的硫醇诱导环化反应生成四氢吲哚衍生物，这是其具有强大抗肿瘤活性的原因。当与蛋白结合时，蛋白会改变环化反应途径，形成独特的羟基异色烯型产物。蛋白如何指导烯二炔化学是一个有趣的难题，过去曾提出过各种建议。我们筛选了五十多种巯基，并操纵条件以确定反应特征并寻找可能影响蛋白导向强度的因素。巯基和氧浓度依赖性研究表明，维持蛋白空间刚性的二硫键可能在改变环化反应途径中发挥关键作用。为了进行直接证明，我们通过用氧原子取代硫原子在两个二硫键处进行突变。圆二色性和二维 NMR 光谱研究表明，突变既没有改变蛋白构象，也没有改变配体相互作用。对巯基诱导的环化反应的分析表明，Cys(37)-Cys(47)的断裂使蛋白几乎完全失去化学导向能力，而 Cys(88)-Cys(93)的断裂仅产生较小的影响。结果表明，结合腔的空间刚性，而不是整个蛋白，在蛋白导向机制中起着重要作用。
