Mechanisms of Enzymic and Hydride Transfers

描述（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，即质子或氢化物转移作为几乎每个酶促反应的一部分而发生，但从或到失活的碳或氧物质的有效转移是非常困难的非酶促完成。二十多年来，我们一直在探索这个问题的蛋白质晶体学，计算方法和定点诱变的组合。沿着这条路，我们已经发展了许多在晶体学、结构生物学和药物发现中普遍使用的技术。我们的研究揭示了以下一般原理：（1）酶催化质子转移依赖于所涉及基团的扰动pKa值。考虑一个催化剂基。似乎这个基团的基本性质增加了，也许是通过将其与溶剂屏蔽，或者通过在其附近放置适当带电的残基，尽管在大多数情况下扰动的机制尚未得到证明。酶还活化底物（即，增加碳酸或含氧酸的酸度）通过两种方式：通过极化，通常通过氢键捐赠给底物氧原子，以及通过过渡态的静电稳定。(2)氢化物转移的酶催化依赖于邻近和取向。辅酶菌株似乎不起主要作用。将底物和/或辅因子与溶剂屏蔽似乎不是必需的，尽管有时确实会发生。极化，例如，金属离子可活化底物，用于氢化物提取和供给，但通常不涉及明显的活化。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结论性答案的问题。问题是：（1）催化性的酸和碱是如何受到蛋白质环境的干扰的？(2)定向涨落（促进振动）在催化中是否起着重要作用？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，结合QM/MM计算，定点诱变加动力学分析，和一种新的方法，用于扰动蛋白质动力学的结合配体的网站远离活性位点。 我们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;这些反应对每个活细胞都是必不可少的，但我们不了解产生这种非凡化学成就的所有因素。我们选择了一类特殊的反应进行研究，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.