Hydrogen Tunneling in Enzyme Reactions

现在有近20个记录的例子，在酶催化的C-H活化过程中的量子效应的作用。从正在进行的酶中H-隧道效应研究中获得的见解包括认识到酶改变了整个反应势垒的形状（即，反应势垒宽度以及高度），并且蛋白质动力学可能在酶催化的键断裂事件中起作用。进一步的研究将集中在建立一个更坚实和更详细的蛋白质动力学和H-隧道之间的关系。几个氢化物转移反应将进行研究，催化的高温乙醇脱氢酶（ht-ADH）的同源性嗜温马肝醇脱氢酶和嗜热二氢叶酸还原酶（ht-DHFR）具有显着的同源性嗜温DHFR。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。这种结构信息将指导位点特异性诱变实验，以分析蛋白质侧链对催化的影响。特别是，在高温下的隧道是占主导地位，并在降低温度下，H-转移变得更加经典的影响将进行分析。 由于ht-ADH太大，无法通过NMR、H/D交换、随后有限的蛋白水解和质谱分析进行动力学研究，因此将进行蛋白质柔韧性图谱的绘制。该实验室最近成功地从B克隆并过表达了ht-DHFR。热脂肪将分析该蛋白质的隧穿对H-转移的贡献作为温度的函数。在平行研究中，将通过NMR（作为局部动力学的探针）和FT-IR H/D交换（作为全局灵活性的探针）研究这种小蛋白质（19 KDa）。

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).