Mechanisms of Enzymic and Hydride Transfers

酶和氢化物转移的机制

• 批准号: 7821369
• 负责人: GREGORY A PETSKO
• 金额: $ 39.04万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-16 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7821369
• 关键词: Achievement; Acidity; Acids; Active Sites; Affinity; Benzaldehyde; Binding; Biochemical Reaction; Biological Models; Carbon; Catalysis; Cells; Charge; Chemicals; Chemistry; Coenzymes; Complex; Computational Technique; Computer Simulation; Crystallography; Data; Dihydrofolate Reductase; Electrostatics; Environment; Enzymes; Glycerol; Goals; Hydrogen; Hydrogen Bonding; Ions; Kinetics; Knowledge; Lead; Learning; Left; Life; Ligand Binding; Measurement; Mechanics; Medical; Metals; Methods; Microscopic; Modeling; Motion; Muscle Rigidity; Mutagenesis; Nature; Neutron Diffraction; Neutrons; Niacinamide; Nitrogen; Oxidoreductase; Oxygen; Peptide Hydrolases; Play; Positioning Attribute; Protein Dynamics; Proteins; Protons; Reaction; Research; Resolution; Roentgen Rays; Role; Site; Site-Directed Mutagenesis; Solvents; Speed; Steroid Isomerases; Structure; System; Techniques; Temperature; Testing; Titrations; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; base; chemical reaction; chymotrypsin; cofactor; design; drug discovery; enzyme substrate; fascinate; galactose mutarotase; molecular dynamics; novel; protein structure; protonation; quantum; research study; simulation; small molecule; stem; structural biology; sugar; ultra high resolution; vibration

DESCRIPTION (provided by applicant): Ever since enzymes were first discovered, the question of how they achieve their remarkable catalytic proficiency has fascinated both chemists and biologists. This project aims to find general answers to this question for a particular class of enzymes: those that catalyze the transfer of a hydrogen ion, either a proton or a hydride, between parts of a substrate and between the enzyme and the substrate. Our focus on hydrogen stems from the fact that proton or hydride transfers occur as a part of nearly every enzymatic reaction, yet efficient transfer from or to an inactivated carbon or oxygen species is very difficult to accomplish non-enzymatically. For over two decades we have probed this question by a combination of protein crystallography, computational approaches, and site-directed mutagenesis. Along the way we have developed a number of techniques of general use in crystallography, structural biology, and drug discovery. Our studies have revealed the following general principles: (1) Enzymatic catalysis of proton transfer depends on perturbed pKa values for the groups involved. Consider a catalytic base. It appears that the basic nature of this group is increased, perhaps either by shielding it from the solvent or by placing an appropriately charged residue near it, although the mechanism of perturbation has not been proven in most cases. Enzymes also activate the substrate (i.e., increase the acidity of the carbon or oxygen acid) in two ways: by polarization, usually by hydrogen bond donation to a substrate oxygen atom, and by electrostatic stabilization of transition states. (2) Enzymatic catalysis of hydride transfer depends on proximity and orientation. Coenzyme strain does not appear to play a major role. Shielding of the substrate and/or cofactor from solvent does not seem to be essential, although it does occur sometimes. Polarization by, e.g., a metal ion can activate a substrate for hydride abstraction and donation, but often there is no obvious activation involved. For this renewal, we wish to focus on a set of questions that we believe have not been answered conclusively by any experiments or calculation. The questions are: (1) How are the catalytic acids and bases perturbed by the protein environment? (2) Do directed fluctuations (promoting vibrations) play a significant role in catalysis? To answer these questions we have selected several enzymes as model systems: four that catalyze proton transfer and three that catalyze both hydride and proton transfers. The specific methods we will employ include ultra-high resolution X-ray crystallography, neutron diffraction, combined QM/MM calculations, site-directed mutagenesis plus kinetic analysis, and a novel method for perturbing protein dynamics by binding ligands to sites remote from the active site. PUBLICE HEALTH RELEVANCE We are trying to understand how the environment of an enzyme makes difficult chemical reactions occur at blinding speed; such reactions are essential for every living cell, yet we don't understand all of the factors that go into producing this extraordinary chemical achievement. We have selected a particular class of reactions for study, and have devised a research plan that makes use of a large number of experimental and computational techniques to dissect what the protein is doing in each case to facilitate the chemistry. If we are successful, the principles we uncover could lead to the design of artificial enzymes for industrial and medical use.

描述（由申请人提供）：自从酶首次被发现以来，它们如何实现其卓越的催化能力的问题一直让化学家和生物学家着迷。该项目旨在为特定类别的酶找到该问题的一般答案：那些催化氢离子（质子或氢化物）在底物部分之间以及酶与底物之间转移的酶。我们对氢的关注源于这样一个事实：质子或氢化物转移几乎是每个酶促反应的一部分，但非酶促地很难实现从或向失活碳或氧物种的有效转移。二十多年来，我们通过结合蛋白质晶体学、计算方法和定点诱变来探索这个问题。一路走来，我们开发了许多晶体学、结构生物学和药物发现领域通用的技术。我们的研究揭示了以下一般原则：（1）质子转移的酶催化取决于所涉及基团的扰动 pKa 值。考虑催化碱。尽管在大多数情况下扰动机制尚未得到证实，但似乎可以通过将其与溶剂隔离或在其附近放置适当带电的残基来增强该基团的基本性质。酶还以两种方式激活底物（即增加碳或含氧酸的酸度）：通过极化，通常通过向底物氧原子提供氢键，以及通过过渡态的静电稳定。 (2)氢化物转移的酶催化取决于接近度和方向。辅酶菌株似乎没有发挥主要作用。将底物和/或辅因子与溶剂隔离似乎并不重要，尽管有时确实会发生。例如，金属离子的极化可以激活用于氢化物提取和供给的底物，但通常不涉及明显的激活。对于本次更新，我们希望重点关注一系列我们认为尚未通过任何实验或计算得出结论性答案的问题。问题是：（1）催化酸和碱如何受到蛋白质环境的干扰？ (2) 定向波动（促进振动）在催化中发挥重要作用吗？为了回答这些问题，我们选择了几种酶作为模型系统：四种催化质子转移，三种同时催化氢化物和质子转移。我们将采用的具体方法包括超高分辨率 X 射线晶体学、中子衍射、组合 QM/MM 计算、定点诱变加动力学分析，以及通过将配体与远离活性位点的位点结合来扰动蛋白质动力学的新方法。 公共健康相关性我们正在试图了解酶的环境如何使困难的化学反应以惊人的速度发生；这些反应对于每个活细胞都至关重要，但我们并不了解产生这一非凡化学成就的所有因素。我们选择了一类特定的反应进行研究，并制定了一项研究计划，利用大量的实验和计算技术来剖析蛋白质在每种情况下的作用，以促进化学反应。如果我们成功了，我们发现的原理可能会导致工业和医疗用途人造酶的设计。