Hydrogen Tunneling in Enzyme Reactions

酶反应中的氢隧道

• 批准号: 0135446
• 负责人: Judith Klinman
• 金额: $ 36万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0135446&HistoricalAwards=false
• 关键词: Hydrogen; Tunneling; Enzyme; Reactions

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).

现在有近 20 个记录在案的例子，说明量子效应在酶催化的 C-H 激活过程中的作用。正在进行的酶中氢通道工作的见解包括认识到酶会改变整个反应势垒形状（即反应势垒宽度和高度），并且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于在蛋白质动力学和氢通道之间建立更牢固、更详细的关系。将研究几种氢化物转移反应，由与嗜温马肝乙醇脱氢酶同源的高温乙醇脱氢酶（ht-ADH）和与嗜温DHFR显着同源的嗜热二氢叶酸还原酶（ht-DHFR）催化。就 ht-ADH 而言，正在研究解决该酶的三维结构。该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，将分析在隧道效应占主导地位的高温下和氢转移变得更加经典的低温下的影响。 由于 ht-ADH 太大，无法通过 NMR 进行动力学研究，因此将进行 H/D 交换，然后进行有限的蛋白水解和质谱分析，以绘制蛋白质的灵活性。该实验室最近成功地从嗜热脂肪芽孢杆菌中克隆并过度表达了 ht-DHFR。将分析该蛋白质作为温度函数的隧道效应对氢传输的贡献。在并行研究中，这种小蛋白质 (19 KDa) 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）进行研究。