Hydrogen Tunneling in Enzyme Reactions

现在有近20个记录的例子表明量子效应在酶催化的碳氢化合物活化过程中所起的作用。从正在进行的酶h隧道研究中获得的见解包括酶修饰整个反应屏障形状（即反应屏障宽度和高度）以及蛋白质动力学可能在酶催化的键裂解事件中发挥作用。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。几个氢化物转移反应将被研究，由高温乙醇脱氢酶（ht-ADH）和嗜热二氢叶酸还原酶（ht-DHFR）催化，高温乙醇脱氢酶（ht-ADH）与嗜温马肝乙醇脱氢酶（ht-DHFR）具有显著的同源性。在ht-ADH的情况下，研究正在进行中，以解决该酶的三维结构。这些结构信息将指导位点特异性诱变实验，分析蛋白质侧链对催化的影响。特别是，在高温下隧穿作用占主导地位，在低温下h转移变得更经典的影响将被分析。由于ht-ADH太大，无法通过NMR进行动力学研究，因此将采用H/D交换，然后进行有限的蛋白质水解和质谱分析来绘制蛋白质的灵活性。该实验室最近成功地克隆并过表达了嗜热脂芽胞杆菌的ht-DHFR。该蛋白将被分析作为温度函数的隧穿对h传递的贡献。在平行研究中，这种小蛋白（19 KDa）将通过NMR（作为局部动力学探针）和FT-IR H/D交换（作为全局灵活性探针）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).