Tunneling and dynamic studies with DHFR

描述：(申请人提供)拟议研究的目标是 为了更好地理解酶如何激活稳定的共价 债券。一种具有生物和医学意义的机械研究 二氢叶酸还原酶(DHFR)将激活C-H键 指挥。我们将研究蛋白质动力学、耦合运动和 催化中的量子力学氢隧道效应。隧道效应是一种现象 粒子通过反应能垒传递，这是由于它的 波浪式属性。多个原子核沿反应的耦合运动 坐标是另一个被发现在某些情况下很重要的现象 酶催化的反应。我们希望解决的主要问题是：是否 这种酶的动力学增强了它所催化的化学作用？ 我们建议研究酶动力学改变对自然界的影响。 它催化的C-H-C转移。隧道和耦合运动将 作为酶复合体中化学步骤的性质的探测器 催化级联。这种酶的动力学将通过定点定向改变 诱变等方法。变化的动力学对自然界的影响 将对H-转移进行检查。我们将应用几种理论模型。 将动力学与隧道和耦合运动的程度相关联。 在这里提出的项目中，将开发一种方法来研究程度 研究了DHFR催化反应中的隧穿和耦合运动。这个 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。 -寻求蛋白质动力学和蛋白质性质之间的关联 H-转接。 拟议中的项目将使我们深入了解 DHFR和生物催化的基本方面。一种方法论 用一种常见的生物还原辅因子研究氢化物隧穿 (烟酰胺)对于研究许多酶是非常有用的。 利用这一辅因。更好地从机理上理解这些酶 可能导致合理设计抑制剂的新方法，并将 促进药物的设计。

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.