Practical Aspects on the Use of Kinetic Isotope Effects as Probes of Flavoprotein Enzyme Mechanisms
Practical Aspects on the Use of Kinetic Isotope Effects as Probes of Flavoprotein Enzyme Mechanisms
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DOI：
10.1007/978-1-4939-0452-5_8
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发表时间：
2014-01-01
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影响因子：
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通讯作者：
Scrutton, Nigel S.
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文献类型：
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作者：
Pudney, Christopher R.;Hay, Sam;Scrutton, Nigel S.
动力学同位素效应（KIEs）的测量已被证明是有用的许多酶活性的机理研究，最显着的是在酶催化的氢转移反应。大于1的初级KIE（1度KIE）表明感兴趣的氢物种的转移是部分或完全速率限制的，并且这些KIE的温度和压力依赖性的大小的研究可以告知转移的机制。例如，KIE测量已经证明对于理解酶系统中量子力学隧穿的作用至关重要。二级KIEs（2度KIEs）的测量也是信息丰富的，可以用来推断一个显着的隧穿贡献和过渡态几何形状的细节。在这里，氘标记物被引入到转移的氢的标记物旁边。1度和2度KIE的测量越来越多地用于黄素蛋白酶中H-转移的研究，这需要制备高纯度和立体特异性标记的同位素体。标记底物的合成策略取决于所研究的酶系统。然而，烟酰胺辅酶常用于黄素蛋白酶机制的研究。在这里，我们提供了酶促合成高纯度氘代同位素体的常见生物辅酶NADH和NADPH以及相应的非反应性模拟物，四氢NAD（P）H的实用细节。这两种形式的辅酶已被证明在机制研究中很有用，特别是与酶促氢转移化学中量子力学隧道和动力学的参与有关。这里的重点是在合成这些化合物的实际考虑。我们还提供了一个简短的描述如何测量KIE可以告知黄素蛋白的机制。这篇文章的目的不是详细描述基本的理论（在文献中已经进行了广泛的综述），而是为那些希望将这种测量纳入酶机制研究的非专业读者提供一个基本的介绍和实际的考虑。
The measurement of kinetic isotope effects (KIEs) has proved useful in many mechanistic studies of enzyme activity, most notably in enzyme-catalyzed hydrogen-transfer reactions. Primary KIEs (1 degrees KIE) greater than unity indicate that transfer of the hydrogen species of interest is partially or fully rate limiting, and studies of the magnitude of the temperature and pressure dependence of these KIEs can inform on the mechanism of transfer. For example, KIE measurements have proved crucial in understanding the role of quantum mechanical tunneling in enzyme systems. The measurement of secondary KIEs (2 degrees KIEs) is also informative and can be used to infer a significant tunneling contribution and details of transition state geometry. Here the deuterium label is introduced next to that of the transferred hydrogen. Measurements of 1 degrees and 2 degrees KIEs are being used increasingly in studies of H-transfer in flavoprotein enzymes and this requires the preparation of high purity and stereospecific labeled isotopologues. Strategies for the synthesis of labeled substrates are dependent on the enzyme system being studied. However, the nicotinamide coenzymes are often used in studies of flavoprotein enzyme mechanisms. Here, we provide practical details for the enzymatic synthesis of high purity deuterated isotopologues of the common biological coenzymes NADH and NADPH as well as the corresponding nonreactive mimics, tetrahydroNAD(P)H. Both forms of the coenzyme have proven useful in the study of mechanisms, particularly in relation to the involvement of quantum mechanical tunneling and dynamics in enzymatic H-transfer chemistry. The focus here is on practical considerations in the synthesis of these compounds. We also provide an abbreviated description of how measurements of KIEs can inform on flavoprotein mechanisms. The aim of this contribution is not to give a detailed description of the underlying theory (which has been reviewed extensively in the literature), but to provide a basic introduction and practical considerations for nonexpert readers who wish to incorporate such measurements in studies of enzyme mechanisms.