Strictosidine synthase:: Mechanism of a Pictet-Spengler catalyzing enzyme
Strictosidine synthase:: Mechanism of a Pictet-Spengler catalyzing enzyme
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DOI：
10.1021/ja077190z
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发表时间：
2008-01-16
影响因子：
15
通讯作者：
O'Connor, Sarah E.
中科院分区：
文献类型：
--
作者：
Maresh, Justin J.;Giddings, Lesley-Ann;O'Connor, Sarah E.
Pictet-Spengler反应是一种由芳香胺和醛生成-碳碱或四氢喹啉的反应，广泛应用于植物生物碱的生物合成。在这里，我们反卷积的作用，生物合成酶缩糖醇合成酶在催化立体选择性合成β -碳碱产物。值得注意的是，通过初级动力学同位素效应（KIE）的出现，酶机制的速率控制步骤是带正电的中间体的重芳构化。非酶Pictet-Spengler反应的KIE表明，溶液中的重芳化也具有速率控制作用，表明酶对反应的机理没有明显的改变。此外，溶液和酶促反应的pH依赖性为催化Pictet-Spengler反应的一系列酸碱催化步骤提供了证据。另外一个酸催化的步骤，很可能是碳胺中间体的质子化，也显著地控制着速率。我们认为这一步是由酶有效催化的。结合酶的双底物抑制剂的结构分析表明，活性位点被精确地调整为正确定向的铝中间体，以产生环化，形成非对映选择性产物。此外，从头算出了该机制中可能的生产过渡态的结构。重要的是，这些计算表明，在picet - spengler机制中经常调用的螺哚林中间体没有发生。根据这些数据提出了酶催化β -碳碱产物的详细机理。
The Pictet-Spengler reaction, which yields either a beta-carboline or a tetrahydroquinoline product from an aromatic amine and an aldehyde, is widely utilized in plant alkaloid biosynthesis. Here we deconvolute the role that the biosynthetic enzyme strictosidine synthase plays in catalyzing the stereoselective synthesis of a beta-carboline product. Notably, the rate-controlling step of the enzyme mechanism, as identified by the appearance of a primary kinetic isotope effect (KIE), is the rearomatization of a positively charged intermediate. The KIE of a nonenzymatic Pictet-Spengler reaction indicates that rearomatization is also rate-controlling in solution, suggesting that the enzyme does not significantly change the mechanism of the reaction. Additionally, the pH dependence of the solution and enzymatic reactions provides evidence for a sequence of acid-base catalysis steps that catalyze the Pictet-Spengler reaction. An additional acid-catalyzed step, most likely protonation of a carbinolamine intermediate, is also significantly rate controlling. We propose that this step is efficiently catalyzed by the enzyme. Structural analysis of a bisubstrate inhibitor bound to the enzyme suggests that the active site is exquisitely tuned to correctly orient the iminium intermediate for productive cyclization to form the diastereoselective product. Furthermore, ab initio calculations suggest the structures of possible productive transition states involved in the mechanism. Importantly, these calculations suggest that a spiroindolenine intermediate, often invoked in the Pictet-Spengler mechanism, does not occur. A detailed mechanism for enzymatic catalysis of the beta-carboline product is proposed from these data.