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中文摘要
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拟议研究的长期目标是更好地了解酶如何稳定地激活 共价键选择性二氢叶酸还原酶中C-H键活化机理的研究 (DHFR)和胸苷酸合成酶(TSase)。这些酶在DNA中起着关键作用 生物合成,并因此用作抗生素和化疗药物的靶标。他们也是模范 系统用于解决酶学中的基本问题,如蛋白质动力学的作用, 环境耦合量子力学氢隧穿键激活。 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蛋白质, 转型,并在上一个赠款期间进行了广泛的研究。种方法 开发用于研究C-H-C转移的物理性质。已经证明,cDHFR 反应坐标是完美安排的H-隧道,远离活性位点的突变体可以 协同干扰H-转移过程。这些发现表明了一个耦合运动网络 通过酶来增强催化反应。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质的影响。 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 将研究复杂TSase催化反应中不同的C-H活化步骤。这些研究将 包括检查控制活性位点突变对不同H- 转移步骤。我们还将研究耦合运动网络的改变对动力学的影响, 使用活性位点远端的突变的动力学。动力学方法的发现可以暴露 特定H-转移步骤的性质将与蛋白质动力学的测量相关,以评估 在快速和限速化学步骤中,运动在增强C-H键活化中的作用。
英文摘要
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.
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Mechanistic Studies of Flavin Dependent Thymidylate Synthase
  • 批准号:
    8787590
  • 项目类别:
  • 资助金额:
    $29.07万
  • 财政年份:
    2014
  • 负责人:
    AMNON KOHEN
  • 依托单位:
Mechanistic studies of nitrogenase catalysis
  • 批准号:
    6629470
  • 项目类别:
  • 资助金额:
    $11.06万
  • 财政年份:
    2002
  • 负责人:
    AMNON KOHEN
  • 依托单位:
Tunneling and dynamic studies with DHFR
  • 批准号:
    6463618
  • 项目类别:
  • 资助金额:
    $17.64万
  • 财政年份:
    2002
  • 负责人:
    AMNON KOHEN
  • 依托单位:
Tunneling and Dynamics in Enzyme Catalyzed Reactions
  • 批准号:
    7724848
  • 项目类别:
  • 资助金额:
    $27.71万
  • 财政年份:
    2002
  • 负责人:
    AMNON KOHEN
  • 依托单位:
海外基金