NOVEL KINETIC TECHNIQUES TO STUDY HYDRATASE REACTIONS

Enoyl-CoA水合酶的反应机理将被深入研究 通过几种新的动力学技术。这些研究的重点是 两个方面：确定酶是否可以将特定的 促进酶-底物结合能转化为催化效率 质子转移反应和确定非共价中间体是否 在催化过程中被水合酶显著稳定 反应。这两个实验目标都需要开发新的 以及更精确的动力学技术，这些技术一般应适用于 其他种类的酶。 考察了巴豆酶动力学参数的pH变化。 确定酶的质子化状态的重要性、影响 底物与活性中心官能团pK-a-S的结合 酶-底物复合体中质子转移的相对速率。二 经过验证的动力学技术，以其精确度而开发，适用于 对酶反应的研究，以提供足够精确的数据。 这些技术应该可以让我们看到Enoyl-CoA水合酶是如何转化的 通过一般的酸碱催化作用将能量结合到催化效率上。 酶结合中间体的存在太不稳定，不能 存在于水溶液中，类似于羧酸的烯醇，是一种 悬而未决的问题。V-max/K-m Will的一系列动力学同位素效应 在其中一个键断裂步骤为Rate的条件下测量 确定建立过渡态的结构。通过 开发重原子和二次氚同位素的方法 V-max可常规测量，相对稳定性不同 将测定酶-底物复合体。 动力学研究提出的中间体的直接证据将 通过在条件下猝灭酶-底物复合体来寻找 这将在化学上区分任何被结合为 二硫代辅酯的反应中间体及其光谱研究 基片模拟。 对酶反应机理的深入了解有助于 在生物科学领域。对人类的作用有了更深入的了解 一般的酸碱催化应改进酶的设计 利用潜在的质子转移的抑制剂，应该 增加对酶活性变化的影响的了解 网站，使我们更接近于设计酶的点 以及想要的活动。

The reaction mechanism of enoyl-CoA hydratase will be intensively studied by several novel kinetic techniques. The emphasis in these studies is two-fold: to determine if enzymes can convert the specific enzyme-substrate binding energy into catalytic efficiency by facilitating proton transfer reactions and to determine if noncovalent intermediates are significantly stabilized by hydratases during the course of the catalyzed reaction. Both of these experimental goals require the development of new and more precise kinetic techniques which should be generally applicable to other classes of enzymes. The pH variation of the kinetic parameters of crotonase will be examined to determine the importance of the protonation state of the enzyme, the effect of substrate binding on the pK-a-s of active site functional groups and the relative rate of proton transfers in the enzyme-substrate complex. Two proven kinetic techniques, developed for their precision, are adapted to the study of enzyme reactions to provide data of sufficient precision. These techniques should allow us to view how enoyl-CoA hydratase converts binding energy to catalytic efficiency through general acid-base catalysis. The presence of enzyme-bound intermediates that would be too unstable to exist in aqueous solution, like the enol of a carboxylic acid, is an unresolved question. A series of kinetic isotope effects on V-max/K-m will be measured under conditions that one of the bond breaking steps is rate determining to establish the structure of the transition state. By developing methods so that heavy atom and secondary deuterium isotope on V-max can be routinely measured, the relative stability of the different enzyme-substrate complexes will be determined. Direct evidence for the intermediates suggested by the kinetic studies will be search for by quenching the enzyme-substrate complex under conditions that will chemically differentiate any of the substrate that was bound as a reactive intermediate and from spectroscopic studies on the dithio-CoAester substrate analog. The enhanced understanding of enzyme reaction mechanisms contributes to all of the biological sciences. The increased understanding of the role of general acid-base catalysis should lead to improved design of enzyme inhibitors that take advantage of the potential proton transfers and should increase the understanding the effects of alterations at enzyme active sites, bringing us a step closer to the point where we can design enzymes with desired activities.