Tunneling and Dynamics in Enzyme Catalyzed Reactions

这项拟议研究的长期目标是更好地了解酶是如何稳定激活的 共价键。二氢叶酸还原酶中C-H键活化的机理研究 将进行DHFRs和胸苷合成酶(TSase)。这些酶在DNA中起着至关重要的作用 因此可作为抗生素和化疗药物的靶标。他们也是模特 用来解决酶学基本问题的系统，如蛋白质动力学和蛋白质动力学的作用 键活化中的环境耦合量子力学氢隧道效应。 目的1：Fola编码的染色体dhfr(Cdhfr)是一种催化单一化学物质的小分子蛋白质。 在上一次赠款期间，已经对这一问题进行了广泛研究。方法： 研究C-H-C转移的物理性质而发展起来的。已经证明，cDHFR 反应坐标被完美地安排用于H隧穿，并且远离活性中心的突变体可以 协同干扰氢转移过程。这些发现表明存在一个耦合运动网络。 通过酶来加强催化反应。拟议的研究将把这些研究扩展到 比较不同的蛋白质支架、动力学和反应物取向对物理性能的影响 C-H-C转移的性质。 目的2：TSase比cDHFR大，催化多个共价键的形成和断裂。我们 将考察络合物TSase催化反应中不同的C-H活化步骤。这些研究将 包括检测受控活性位点突变对不同H-的动力学和动力学影响 转移步骤。我们还将研究改变的耦合运动网络对动力学和 利用活性部位远端突变的动力学。来自动力学方法的发现可以揭示 特定的氢转移步骤的性质将与蛋白质动力学的测量相关联，以评估 在快速和限速化学步骤中，运动在增强C-H键激活中的作用。

The long-term objective of the proposed research is to better understand how enzymes activate stable covalent bonds. A mechanistic investigation of C-H bond activation in alternative dihydrofolate reductases (DHFRs) and thymidylate synthase (TSase) will be conducted. These enzymes play crucial roles in DNA biosynthesis and thus serve as targets for antibiotic and chemotherapeutic drugs. They are also model systems used to address fundamental issues in enzymology, such as the role of protein dynamics and environmentally coupled quantum mechanical hydrogen tunneling in bond activation. Aim 1: The FolA encoded chromosomal DHFR (cDHFR) is a small protein that catalyzes a single chemical transformation and has been studied extensively during the previous grant period. Methods were developed to study the physical nature of the C-H-C transfer. It has been demonstrated that the cDHFR reaction coordinate is perfectly arranged for H-tunneling, and that mutants far from the active site can synergistically disturb the H-transfer process. These findings suggested a network of coupled motions across the enzyme that enhance the catalyzed reaction. The proposed studies will extend these studies to compare the effects of different protein scaffolds, dynamics, and reactants orientation on the physical nature of the C-H-C transfer. Aim 2: TSase is larger than cDHFR and catalyzes the making and breaking of multiple covalent bonds. We will examine different C-H activation steps in the complex TSase catalyzed reaction. These studies will include the examination of kinetic and dynamic effects of controlled active site mutations on different H- transfer steps. We will also examine the effect of altered networks of coupled motions on kinetics and dynamics using mutations distal to the active site. The findings from kinetic methods that can expose the nature of specific H-transfer steps will be correlated to measurements of protein dynamics, to assess the role of motions in enhancing C-H bond activation, in both fast and rate-limiting chemical steps.