Tunneling and Dynamics in Enzyme Catalyzed Reactions

描述（由申请人提供）：拟议研究的长期目标是更好地了解酶如何激活稳定的共价键。将对二氢叶酸还原酶（DHFRs）和胸苷酸合成酶（TSase）中C-H键激活的机制进行研究。这些酶在DNA生物合成中起着至关重要的作用，因此是抗生素和化疗药物的靶点。它们也是用于解决酶学基本问题的模型系统，例如蛋白质动力学和环境耦合量子力学氢隧道在键激活中的作用。目标1：叶酸编码的染色体DHFR （cDHFR）是一种催化单一化学转化的小蛋白质，在之前的资助期间已被广泛研究。研究了C-H-C转移的物理性质。研究表明，cDHFR反应坐标为h隧穿提供了完美的安排，远离活性位点的突变体可以协同干扰h转移过程。这些发现表明，通过酶的耦合运动网络增强了催化反应。拟议的研究将扩展这些研究，以比较不同蛋白质支架、动力学和反应物取向对C-H-C转移物理性质的影响。目的2:TSase比cDHFR大，催化多个共价键的形成和断裂。我们将研究在复杂的TSase催化反应中不同的C-H活化步骤。这些研究将包括受控活性位点突变对不同H转移步骤的动力学和动力学影响的检查。我们还将研究使用活性位点远端突变改变的耦合运动网络对动力学和动力学的影响。动力学方法的发现可以揭示特定h转移步骤的性质，将与蛋白质动力学的测量相关联，以评估运动在增强C-H键激活中的作用，在快速和限速化学步骤中。公共卫生相关性：将研究对DNA生物合成至关重要的两个酶家族，因此也是抗生素和化疗药物的靶点。这项研究的目的是更好地了解酶的动力学是如何影响它们催化的化学反应的。将蛋白质动力学纳入药物设计的潜在影响是深远的，并可能促进在以组合方法为主的领域进行合理设计的实践。

DESCRIPTION (provided by applicant): 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 funding 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. PUBLIC HEALTH RELEVANCE: Two families of enzymes that are essential for DNA biosynthesis, and hence targets for antibiotic and chemotherapeutic drugs, will be studied. The investigation aims for a better understanding of how the dynamics of enzymes affect the chemistry they catalyze. The potential impact of including protein dynamics in drug design is far-reaching, and may boost practice of rational design in a field dominated by combinatorial approaches.