Tunneling and Dynamics in Enzyme Catalyzed Reactions

描述(由申请人提供)：将通过研究催化单个C-H键激活的小酶来更好地了解酶如何激活共价键，这些研究将扩展到催化单个活性部位内复杂的共价键激活的更大的酶。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这一过程中的作用。这些研究将阐明增强键活性的进化进展，尽管事实上催化剂的周转通常受到化学转化以外的过程的速度限制。目标1将遵循二氢叶酸还原酶(DHFR)从细菌到人类，从DHFR到二氢生物蝶呤还原酶(DHPR)定向进化的化学步骤的本质。目的2将研究活性中心残基在胸苷合成酶(TSase)催化的不同化学转化中的作用，并对计算提出的另一种反应机理进行实验验证。目标3将研究整个蛋白质的化学步骤和快速平衡动力学(飞秒-纳秒)之间的关系。目标4将通过同位素重蛋白(Born-Oppenheimer酶)诱导这些快速动力学的最小扰动，并将探索其对催化化学步骤的结果影响。这种全面的研究将需要大量的实验和理论工具，包括动力学同位素效应的测量和计算；蛋白质结晶学和X射线衍射的各向异性B因子的测量；核磁共振弛豫测量、QM/MM混合计算；振动光谱(2D-IR)；以及定向进化。因此，研究小组由四名分包人、另外三名合作调查员和PI组成。

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.