Tunneling and Dynamics in Enzyme Catalyzed Reactions

描述（由申请方提供）：通过研究催化单个C-H键活化的小酶，将更好地理解酶如何活化共价键，这些研究将扩展到催化单个活性位点内共价键活化的复杂级联的较大酶。这些研究旨在揭示化学步骤（键激活）的性质，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活化的进化进展，尽管事实上，催化营业额通常是速率限制的过程以外的化学转化。 提出了四个具体目标：目标1将遵循化学步骤的本质，沿着二氢叶酸还原酶（DHFR）从细菌到人类的自然进化，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不同的化学转化催化的酶胸苷酸合成酶（TSase）的作用，并将测试实验提出的计算替代反应机制。目的3研究化学步骤与整个蛋白质的快速平衡动力学（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，并将探索对催化化学步骤的影响。 这种全面的研究将需要广泛的实验和理论工具，包括动力学同位素效应（KIE）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; NMR弛豫测量，混合QM/MM计算;振动光谱（2D-IR）;和定向进化。因此，研究团队由4名分包商、3名其他合作研究者和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.