STRUCTURES INFLUENCE ON REACTIVITY IN METALLOENZYMES

描述：本提案中描述的研究将针对： 了解结构如何影响功能和/或特性 金属酶活性中心的性质。了解蛋白质是如何 约束可以影响反应性，也可以影响几何/电子 金属酶的结构。 更具体地说：研究将继续集中在设计和 氰基水合酶铁和钴中心类似物的合成。 具有各种结构的合成模型将被合成 (使用通用的一锅合成法)，结构表征(使用 X-ra结晶学)，然后筛选关键的生物物理特性 如(1)a S=1/2自旋态(EPR，磁性(溶液和固态))， (2)700 nm附近有一个强烈的可见光带。 我们的合成模型与酶底物(RCN)、抑制剂之间的反应 (N3-)和灭活剂(NO)，然后将同时监测 分光光度计法和EPR法。 通过系统地改变我们模型的结构，并探索 这些变化对电子和反应性的影响， 属性，我们将确定结构之间是否存在任何关联 反应性，并确定自旋态和其他电子性质 对反应性有重要影响。 配基约束将被合并到我们的模型中，作为 蛋白质限制，这样我们就可以探索蛋白质的影响 约束可以对结构和反应性产生影响。例如，通过使用 具有固定“主链”长度的配体，角度可以被约束，并且，在 有些情况下，打开后，就会形成一个承印物结合部位(内位态)。 类似的限制可能是导致金属酶活性的原因。 旨在了解其他化合物的结构/活性关系的研究 半胱氨酸连接的金属酶，如氢酶和肝醇 脱氢酶，也将被追求。

DESCRIPTION: Research described in this proposal will be aimed at: Understanding how structure influences function and/or the characteristic properties of metalloenzyme active sites. Understanding how protein constraint can influence both the reactivity, and geometric/electronic structure of metalloenzymes. More specifically: Studies will continue to focus on the design and synthesis of analogues for the iron and cobalt sites of nitrile hydratases. Synthetic models, possessing a variety of structures, will be synthesized (using a versatile, one-pot synthesis), structurally characterized (using X-ra crystallography), and then screened for key biophysical properties such as (1) a S=1/2 spin-state (epr, magnetism (both solution and solid state)), and (2) a intense visible band near 700 nm. Reactions between our synthetic models and enzyme substrates (RCN),inhibitor (N3-), and inactivator (NO), will then be monitored both spectrophotometricall and by EPR. By systematically altering the structure of our models, and probing the influence that these changes have on the electronic, and reactivity, properties, we will determine if there is any correlation between structure an reactivity, and determine if spin-state and other electronic properties have a important influence on reactivity. Ligand constraints will be incorporated into our models, as a model for protei constraints, so that we can probe the influence that protein constraints can have on structure and reactivity. For example, by using a ligand with a fixed "backbone" length, angles can be constrained, and, in some cases, opened, so a to create a substrate binding site (entatic state). Similar constraints may be responsible for the reactivity of metalloenzymes. Studies aimed at understanding structure/reactivity relationships in other cysteine-ligated metalloenzymes, such as hydrogenase, and liver alcohol dehydrogenase, will also be pursued.