Hydrogen Tunneling in Enzyme Reactions

现在有近20个记录在案的例子说明了量子效应在酶催化的C-H激活过程中的作用。正在进行的关于酶中H隧道作用的工作包括认识到酶改变了整个反应屏障的形状(即反应屏障的宽度和高度)，蛋白质动力学可能在酶催化的键断裂事件中发挥作用。进一步的研究将集中在建立蛋白质动力学和H隧穿之间更牢固和更详细的关系上。几个氢化物转移反应将被研究，催化的高温醇脱氢酶(Ht-ADH)与中温马肝醇脱氢酶(Ht-ADH)和高温二氢叶酸还原酶(Ht-DHFR)与中温DHFR有显著同源性。在HT-ADH的情况下，研究正在进行中，以解决这种酶的三维结构。这些结构信息将指导定点突变实验，以分析蛋白质侧链对催化的影响。特别是，将分析在隧道效应占主导地位的高温和氢转移变得更加经典的温度降低时的影响。由于Ht-ADH太大，不能用核磁共振研究动力学，因此将进行H/D交换，然后进行有限的蛋白质分解和质谱分析，以确定蛋白质的柔韧性。本实验室最近成功地克隆并高效表达了嗜热脂肪芽孢杆菌的hT-dhfr基因。将分析该蛋白质的隧道效应对氢转移的贡献，作为温度的函数。在平行研究中，这种小蛋白(19 KDa)将通过核磁共振(作为局部动力学的探针)和FT-IR H/D交换(作为全球灵活性的探针)进行研究。

There are now close to 20 documented examples of a role for quantum effects in enzyme catalyzed C-H activation processes. Insights from the ongoing work on H-tunneling in enzymes include the recognition that enzymes modify the entire reaction barrier shape (i.e., reaction barrier width as well as height) and that protein dynamics may play a role in the bond cleavage events catalyzed by enzymes. Further investigations are to be focused on establishing a firmer and more detailed relationship between protein dynamics and H-tunneling. Several hydride transfer reactions will be studied, catalyzed by a high temperature alcohol dehydrogenase (ht-ADH) with homology to the mesophilic horse liver alcohol dehydrogenase and a thermophilic dihydrofolate reductase (ht-DHFR) with significant homology to mesophilic DHFR. In the case of ht-ADH, studies are in progress to solve the three dimensional structure for this enzyme. This structural information will guide site specific mutagenesis experiments to analyze the effect of protein sidechains on catalysis. In particular, the effects at elevated temperatures where tunneling is dominant and at reduced temperatures where H-transfer becomes more classical will be analyzed. Since the ht-ADH is too large for a study of dynamics by NMR, H/D exchange, followed by limited proteolysis and mass spectrometric analysis will be pursued to map out protein flexibility. This laboratory has recently succeeded in cloning and over-expressing a ht-DHFR from B. stearothermophilis. This protein will be analyzed for the contribution of tunneling to H-transfer as a function of temperature. In parallel studies, this small protein (19 KDa) will be studied by NMR (as a probe of local dynamics) and by FT-IR H/D exchange (as a probe of global flexibility).