Hydrogen Tunneling in Enzyme Reactions

酶反应中的氢隧道

• 批准号: 9514126
• 负责人: Judith Klinman
• 金额: $ 30万
• 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: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 1999-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9514126&HistoricalAwards=false
• 关键词: Hydrogen; Tunneling; Enzyme; Reactions

9507018 Klinman The original observations of room-temperature hydrogen tunneling in enzyme reactions occured in the yeast alcohol dehydrogenase and the bovine serum amine oxidase reactions. In recent years, evidence for tunneling has been exteneded to include horse liver alcohol dehudrogenase, monoamine oxidase, and very likely soybean lipoxygenase and glucose oxidase. When conditions are optimized to allow the monitoring of the hydrogen transfer step itself, it appears that the phenomenon of tunneling can be readily detected, although the exact nature of tunneling varies from one system to another. Our findings suggest that, in conjuction with the more classical sources of catalytic rate enhancement, the structures at enzyme active sites have been optimized to make catalytic use of quantum effects. One of the most extreme types of behavior has been seen in the soybean lipoxgenase reaction, which is postulated to catalyze substrate oxidation by close to full quantum event. Future studies with this enzyme will focus on (i) the use of stopped flow kinetics to monitor the H-transfer step directly as function of temperature; (ii) the kinetic characterization of D/T and H/T labeled substrates as probes of tunneling. Premiliary studies of D/T and H/T isotope effects with two forms of glucose oxidase, which differ ca. two-fold in mass (due solely to differences in patterns of glycosylation),suggest different degrees of tunneling. These proteins will be deglycosylated and recharacterized to test the hypothesis that overall protein mass (and possibly mobility) can influence tnneling. The horse liver alcohol dehydrogenase offers a superb opportunity to examine the role of specific active site side chains in optimizing tunneling. A systematic study of residues contacting the bound cofactor is planned by site specific mutagenesis. In order or pursue the question of a possible link between protein dynamics and efficient hydrogen tunneling, two experimental routes wi ll be taken. These involve (I) the study of horse liver alcohol dehydrogenase at low temperature in cryosolvent using a photodisociable, substrate precursor and (ii) the study of D/T and H/T isotope effects as a function of temperture (between ca. 25 and 90oC) using thermophilic forms of alcohol dehydrogenase. Brief Description (200 words) of Project The accepted paradigm for the enormous rate accelerations brought about by protein catalysts (enzymes) has been a reduction in the height of the energy barrier for the conversion of reactant to product. Quantum mechanics provides a more fundamental and general way of formalizing reaction dyamics. However, with the exception of electron transfer reactions, it has generally been assumed that proteins, because of their large size, catalyze reactions classically. In fact, we have recently shown that under physiologic conditions, quantum effects contribute significantly to the tate accelerations brought about by many enzymes. We are now trying to understand the specific ways in which enzymes take advantage of quantum mechanics to enhance their catalytic effects. Three areas are under investigation: these include roles of specific amino acid side chain interactions, overall protein size, and protein dynamics in the manifestation of catalysis through quantum effects.

酶反应中的室温氢隧穿现象最早见于酵母醇脱氢酶和牛血清胺氧化酶反应中。近年来，隧道效应的证据已经扩展到包括马肝酒精脱氢酶，单胺氧化酶，很可能还有大豆脂氧化酶和葡萄糖氧化酶。当条件优化到允许监测氢转移步骤本身时，隧道现象似乎可以很容易地检测到，尽管隧道的确切性质因系统而异。我们的研究结果表明，结合更经典的催化速率增强来源，酶活性位点的结构已被优化，以利用量子效应进行催化。在大豆脂氧化酶反应中已经看到了一种最极端的行为类型，该反应被假定为通过接近全量子事件催化底物氧化。该酶的未来研究将集中在(i)使用停止流动动力学直接监测h转移步骤作为温度的函数；（ii） D/T和H/T标记基质作为隧道探针的动力学表征。对两种形式的葡萄糖氧化酶的D/T和H/T同位素效应的初步研究表明，两种形式的葡萄糖氧化酶在质量上相差约两倍（仅仅是由于糖基化模式的差异），存在不同程度的隧道效应。这些蛋白质将被去糖基化并重新表征，以测试总蛋白质质量（可能还有流动性）会影响穿洞的假设。马肝酒精脱氢酶提供了一个极好的机会来研究特定活性位点侧链在优化隧道中的作用。系统地研究与结合辅因子接触的残基是通过位点特异性诱变计划的。为了探究蛋白质动力学和有效氢隧穿之间的可能联系，我们将采取两条实验路线。这包括(I)在低温溶剂中使用可光降解的底物前体研究马肝酒精脱氢酶，以及（ii）使用嗜热形式的酒精脱氢酶研究D/T和H/T同位素效应作为温度（约25至90摄氏度）的函数。对于蛋白质催化剂（酶）带来的巨大速率加速，公认的范式是降低了反应物转化为产物的能量垒的高度。量子力学为反应动力学的形式化提供了一种更为基本和普遍的方法。然而，除了电子转移反应外，人们通常认为，由于蛋白质的体积大，它们通常催化反应。事实上，我们最近已经证明，在生理条件下，量子效应对许多酶带来的状态加速起着重要作用。我们现在正试图了解酶利用量子力学来增强其催化作用的具体方式。三个领域正在研究中：这些包括特定氨基酸侧链相互作用的作用，整体蛋白质大小，以及通过量子效应催化表现的蛋白质动力学。