Pudney Christopher R, Hay Sam, Scrutton Nigel S
Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
Methods Mol Biol. 2014;1146:161-75. doi: 10.1007/978-1-4939-0452-5_8.
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° 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° 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° and 2° 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.
事实证明，动力学同位素效应（KIEs）的测量在酶活性的许多机理研究中都很有用，最显著的是在酶催化的氢转移反应中。大于1的一级KIEs（1°KIE）表明目标氢物种的转移部分或完全是速率限制因素，对这些KIEs的温度和压力依赖性大小的研究可以为转移机制提供信息。例如，KIE测量已被证明对于理解量子力学隧穿在酶系统中的作用至关重要。二级KIEs（2°KIEs）的测量也具有参考价值，可用于推断显著的隧穿贡献和过渡态几何结构的细节。此处，氘标记被引入到转移氢的旁边。1°和2°KIEs的测量在黄素蛋白酶的氢转移研究中越来越多地被使用，这需要制备高纯度和立体特异性标记的同位素类似物。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。在此，我们提供了关于常见生物辅酶NADH和NADPH以及相应的非反应性模拟物四氢NAD(P)H的高纯度氘代同位素类似物酶促合成的实用细节。这两种形式的辅酶已被证明在机制研究中很有用，特别是在酶促氢转移化学中量子力学隧穿和动力学的参与方面。这里的重点是这些化合物合成中的实际考虑因素。我们还简要描述了KIEs测量如何为黄素蛋白酶机制提供信息。本论文的目的不是详细描述基础理论（该理论在文献中已有广泛综述），而是为希望将此类测量纳入酶机制研究的非专业读者提供基本介绍和实际考虑因素。
