Mechanisms of Enzymic and Hydride Transfers

描述(申请人提供)：自从酶被首次发现以来，它们如何实现其非凡的催化熟练程度的问题一直吸引着化学家和生物学家。这个项目旨在为一类特殊的酶找到这个问题的一般答案：那些催化氢离子转移的酶，无论是质子还是氢化物，在底物的各个部分之间以及酶和底物之间。我们对氢的关注源于这样一个事实，即质子或氢化物的转移几乎是每个酶反应的一部分，然而，从失活的碳或氧物种进行有效的转移是很难在非酶作用下完成的。二十多年来，我们通过蛋白质结晶学、计算方法和定点突变的组合来探索这个问题。在这一过程中，我们开发了许多在结晶学、结构生物学和药物发现方面普遍使用的技术。我们的研究揭示了以下一般规律：(1)质子转移的酶催化依赖于所涉及基团的微扰pKa值。考虑一种催化碱。它似乎增加了这个基团的基本性质，可能是通过使其远离溶剂或通过在其附近放置适当的带电残基，尽管在大多数情况下尚未证明微扰的机制。酶还以两种方式激活底物(即增加碳酸或氧酸的酸度)：通过极化，通常通过氢键给予底物氧原子，以及通过静电稳定过渡态。(2)酶催化氢化物转移反应依赖于亲和度和取向。辅酶菌株似乎没有发挥主要作用。底物和/或辅因子对溶剂的屏蔽似乎不是必不可少的，尽管有时确实会发生这种情况。例如，金属离子的极化可以激活底物以进行氢化物提取和捐赠，但通常不涉及明显的激活。对于这次更新，我们希望集中讨论一系列我们认为尚未通过任何实验或计算得出最终答案的问题。问题是：(1)催化酸和碱是如何被蛋白质环境扰动的？(2)定向波动(促进振动)在催化中起重要作用吗？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。我们将使用的具体方法包括超高分辨率X射线结晶学、中子衍射、QM/MM组合计算、定点突变和动力学分析，以及一种通过将配体结合到远离活性中心的位置来扰动蛋白质动力学的新方法。与公众健康相关我们正试图了解酶的环境如何使困难的化学反应以令人眼花缭乱的速度发生；这种反应对每个活细胞都是必不可少的，但我们并不了解导致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都在做什么，以促进化学。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人造酶的设计。

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.