Mechanisms of Enzymic and Hydride Transfers

描述（由申请人提供）：自从酶第一次被发现以来，它们是如何实现其非凡的催化能力的问题一直吸引着化学家和生物学家。该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（质子或氢化物）在底物部分之间以及酶和底物之间转移的酶。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一个酶促反应的一部分，但从一个灭活的碳或氧物种之间有效的转移是非常困难的非酶完成。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。在此过程中，我们开发了许多在晶体学、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原理：(1)质子转移的酶催化依赖于所涉及基团的扰动pKa值。考虑催化碱。似乎这个基团的基本性质增加了，可能是通过保护它不受溶剂的影响，或者通过在它附近放置一个适当带电的残基，尽管在大多数情况下，扰动的机制尚未得到证实。酶还通过两种方式激活底物（即增加碳或氧酸的酸度）：通过极化，通常是通过氢键给底物氧原子，以及通过过渡态的静电稳定。(2)氢化物转移的酶催化作用取决于邻近性和取向。辅酶菌株似乎不起主要作用。基材和/或辅因子对溶剂的屏蔽似乎不是必需的，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的提取和给予，但通常没有明显的激活。在这次更新中，我们希望把重点放在我们认为没有通过任何实验或计算得到最终回答的一系列问题上。问题是：(1)催化酸和碱是如何受到蛋白质环境的干扰的？(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，三种既催化氢化物转移又催化质子转移。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、QM/MM联合计算、位点定向诱变加动力学分析，以及一种通过将配体结合到远离活性位点的位点来干扰蛋白质动力学的新方法。我们正试图了解一种酶的环境是如何使困难的化学反应以令人眼花缭乱的速度发生的；这样的反应对每个活细胞来说都是必不可少的，然而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，并设计了一个研究计划，利用大量的实验和计算技术来分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的人工酶的设计。

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.