Tunneling and dynamic studies with DHFR

使用 DHFR 进行隧道和动态研究

• 批准号: 6623158
• 负责人: AMNON KOHEN
• 金额: $ 17.7万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6623158
• 关键词: NAD(H) phosphate; X ray crystallography; chemical kinetics; chemical synthesis; computer simulation; covalent bond; dihydrofolate reductase; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate complex; hydrogen; intermolecular interaction; model design /development; molecular dynamics; molecular site; nuclear magnetic resonance spectroscopy; protein purification; quantum chemistry; radiotracer; site directed mutagenesis; temperature; thermostability

DESCRIPTION: (provided by applicant) The objective of the proposed research is to develop a better understanding of how enzymes activate stable covalent bonds. A mechanistic investigation of biologically and medically significant C-H bond activation by the enzyme dihydrofolate reductase (DHFR) will be conducted. We will examine the role of protein dynamics, coupled motion, and quantum mechanical hydrogen tunneling in catalysis. Tunneling is the phenomenon by which a particle transfers through a reaction energy barrier due to its wave-like property. Coupled motion of several nuclei along the reaction coordinate is another phenomenon that has been found to be important in some enzyme catalyzed reactions. The principal problem we wish to address is: does the enzyme's dynamics enhance the chemistry it catalyzes? We propose to investigate the effect of altered enzyme dynamics on the nature of the C-H-C transfer that it catalyzes. Tunneling and coupled motion will serve as probes for the nature of the chemical step in the enzyme complex catalytic cascade. The enzyme's dynamics will be altered by site-directed mutagenesis and other methods. The effect of the altered dynamics on the nature of the H-transfer will be examined. Several theoretical models will be applied to correlate the dynamics to the degree of tunneling and coupled motion. In the project proposed herein, a method will be developed to study the degree of tunneling and coupled motion in the DHFR catalyzed reaction. The experimental design includes: - Measuring the ratio of reaction rates with substrates labeled with the three isotopes of hydrogen (H/D/T kinetic isotope effects) and their temperature dependence. - Data analysis using non-classical methods, which will afford an estimation of the degree of tunneling and coupled motion in the hydride transfer. - Pursuing a correlation between the protein dynamics and the nature of H-transfer. The proposed project will lead to significant insight into the mechanism of DHFR and fundamental aspects of biocatalysis in general. A methodology for looking into hydride tunneling with a common biological reductive cofactor (nicotinamide) would be extremely useful for studying the many enzymes utilizing this cofactor. A better mechanistic understanding of these enzymes could lead to new approaches to the rational design of inhibitors and would facilitate the design of drugs.

描述：（由申请人提供）拟议研究的目的是 为了更好地理解酶如何激活稳定的共价键， 债券一项具有生物学和医学意义的 通过二氢叶酸还原酶（DHFR）的C-H键活化将是 进行。我们将研究蛋白质动力学，耦合运动， 催化中的量子力学氢隧穿。Tunnel是一种现象， 粒子通过反应能量势垒转移， 类波性质多核沿着反应的耦合运动 坐标是另一种现象，已经发现在一些实施例中是重要的。 酶催化反应我们要解决的主要问题是： 酶的动力学增强了它催化的化学反应 我们建议研究改变酶动力学对性质的影响， 它所催化的碳氢碳转移调谐和耦合运动将 用作酶复合物中化学步骤性质的探针 催化级联酶的动力学将被定点 诱变和其他方法。改变的动力学对自然的影响 将对H-转移进行检查。几个理论模型将被应用 将动力学与隧道效应和耦合运动的程度相关联。 在本文提出的项目中，将开发一种方法来研究程度 隧道和耦合运动的DHFR催化反应。的 实验设计包括： - 用三种标记的底物测量反应速率的比率 氢的同位素（H/D/T动力学同位素效应）及其温度 依赖 - 使用非经典方法进行数据分析，这将提供 氢化物转移中的隧穿和耦合运动的程度。 - 追求蛋白质动力学和蛋白质性质之间的相关性， H-转移。 拟议的项目将使人们深入了解 DHFR和一般生物催化的基本方面。一套办法以供 寻找氢化物隧道与一个共同的生物还原辅因子 （烟酰胺）将是非常有用的研究许多酶 利用这个辅助因子。更好地理解这些酶的机制 可能导致抑制剂的合理设计的新方法， 有助于药物的设计。