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Tunneling and Dynamics in Enzyme Catalyzed Reactions

酶催化反应中的隧道效应和动力学

基本信息

批准号：
9225208
负责人：
CHRISTOPHER M CHEATUM
金额：
$ 36.12万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-07-01 至 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9225208
关键词：
Active Sites Antibiotics Bacteria Biomimetics Carbon Chemical Evolution Chemicals Cleaved cell Complex Crystallography Dihydrofolate Reductase Directed Molecular Evolution Drug Design Drug resistance Enzymes Equilibrium Escherichia coli Evolution Factor X Gases Grant Human Hybrids Hydrogen Hydrogen Bonding Investigation Isotopes Kinetics Label Lactate Dehydrogenase Light Measurement Mechanics Methods Molecular Molecular Conformation Motion Mutation Nature Outcome Oxidoreductase Pharmaceutical Preparations Phase Phylogenetic Analysis Process Protein Dynamics Proteins Protons Reaction Relaxation Research Research Personnel Role Series Site-Directed Mutagenesis Spectrum Analysis Structure Substrate Specificity Suggestion System Terminology Testing Thymidylate Synthase Vertebral column X ray diffraction analysis X-Ray Diffraction catalyst covalent bond design experimental study inhibitor/antagonist interest mechanical behavior nanosecond particle protein structure public health relevance quantum response tool vibration

项目摘要

DESCRIPTION (provided by applicant): A better understanding of how enzymes activate covalent bonds will be pursued via the investigation of a small enzyme that catalyzes a single C-H bond activation, and these studies will be extended to a larger enzyme that catalyzes a complex cascade of covalent bond activations within a single active site. The studies aim to reveal the nature of the chemical step (bond activation), and the role of the whole protein structure and dynamics in that process. The studies will illuminate the evolutionary progressions that enhance the bond activation despite the fact that the catalytic turnover is usually rate-limited by processes other than the chemical transformation. Four specific aims are proposed: Aim 1 will follow the nature of the chemical step along the natural evolution of dihydrofolate reductase (DHFR) from bacteria to human, and from DHFR toward dihydrobiopterin reductase (DHPR) by means of directed evolution. Aim 2 will examine the role of active site residues in different chemical conversions catalyzed by the enzyme thymidylate synthase (TSase), and will test experimentally an alternative reaction mechanism proposed by calculations. Aim 3 will study the relations between the chemical step and fast equilibrium dynamics (femtosecond-nanosecond) across the whole protein. Aim 4 will induce a minimal perturbation of those fast dynamics by means of isotopically heavy proteins (Born-Oppenheimer enzymes), and will explore the resultant effects on the catalyzed chemical step. Such comprehensive studies will require a broad arsenal of experimental and theoretical tools including measurements and calculations of kinetic isotope effects (KIEs); protein crystallography and measurements of anisotropic B-factors from X-ray diffractions; NMR relaxation measurements, hybrid QM/MM calculations; vibrational spectroscopy (2D-IR); and directed evolution. Accordingly, the research team is composed of fours subcontractors, three other co-investigators, and the PI.

描述（由申请人提供）：通过对催化单个C-H键激活的小酶的研究，将更好地了解酶如何激活共价键，这些研究将扩展到在单个活性位点内催化复杂级联共价键激活的更大酶。这些研究旨在揭示化学步骤（键激活）的本质，以及整个蛋白质结构和动力学在该过程中的作用。这些研究将阐明增强键激活的进化过程，尽管催化周转通常受到除化学转化以外的过程的速率限制。提出了四个具体目标：目标1将沿着二氢叶酸还原酶（DHFR）从细菌到人类的自然进化的化学步骤的性质，以及通过定向进化从DHFR到二氢生物蝶呤还原酶（DHPR）。目的2将研究活性位点残基在由胸苷酸合成酶（TSase）催化的不同化学转化中的作用，并将通过实验测试计算提出的替代反应机制。目的3将研究整个蛋白质的化学步骤与快速平衡动力学（飞秒-纳秒）之间的关系。目的4将通过同位素重蛋白（Born-Oppenheimer酶）诱导对这些快速动力学的最小扰动，并将探索对催化化学步骤的最终影响。这种全面的研究将需要广泛的实验和理论工具，包括动能同位素效应的测量和计算；蛋白质晶体学和x射线衍射各向异性b因子的测量；核磁共振弛豫测量，混合QM/MM计算；振动光谱（2D-IR）；定向进化。因此，研究小组由4名分包商和3名共同调查人员以及PI组成。