REACTIVE INTERMEDIATES OF ENZYMATIC REACTIONS

描述（由申请人提供）：酶催化的标志是酶对其过渡态的高亲和力，但这种紧密结合的起源通常不太清楚。本申请描述了探索各种酶利用底物磷酸二氢离子基团的大固有结合能来稳定过渡态以形成不稳定碳中间体的机制的实验。我们的中心假设是，灵活的“磷酸盐夹持器”环是广泛分布的保守蛋白质元件，可提供结合能，用于稳定过渡态，形成酶结合碳离子。感兴趣的酶促反应包括醛酮异构化，糖外映异构化，醛醇缩合和脱羧。我们将使用我们的“两部分底物”方案，其中底物磷酸二钠基团，由外源性亚磷酸二钠模拟，与进行化学反应的底物部分分离。通过观察到外源亚磷酸二钠离子对酶催化第二底物片段反应的激活作用，可以量化磷酸二钠离子结合相互作用的特定过渡态稳定性。描述了四个项目，以检查由柔性环-亚磷酸酯相互作用引起的过渡状态稳定的一般性和机制。(1)我们将研究奥罗替丁5'-单磷酸脱羧酶（OMPDC）超家族的成员。目的是确定这些酶的结构保守的磷酸爪环是否具有提供碳离子中间体特异性稳定的共同功能。(2)我们将研究磷酸爪环的长度与酶-磷酸二氢离子结合相互作用之间的关系，这两种酶催化长度仅相差一个碳原子的磷酸化糖的外映体化。(3)我们将探索OMPDC在化学上难以实现的奥罗替丁5'-单磷酸脱羧过程中实现1017倍速率加速的机制。我们将探讨该酶与底物磷酸二氢离子之间的相互作用在稳定远端嘧啶环脱羧过渡态中的作用机制，并解决有关该酶的神秘作用机制的其他问题。(4)我们将继续研究柔性环-磷酸二氢离子相互作用在稳定由磷酸三糖异构酶催化的磷酸三糖醛糖-酮糖异构化过程中烯二醇（酸）中间体形成过渡态中的作用。一个主要的目标是提供一个完整的物理机制的描述，通过这种机制，柔性催化环的运动作为一个“开关”，打开稳定的过渡态相互作用。公共卫生相关性：酶催化剂是所有生命系统的主要组成部分之一，有许多疾病是由单一酶的功能障碍或缺乏引起的。从酶和非酶反应的机制研究中对酶催化的理解的进展可能对药物设计、对代谢途径和疾病的理解以及对其他健康相关问题的解决至关重要。这项应用的重点是灵活的磷酸爪环在酶催化中的关键作用，结果可能会刺激开发专门针对这些环的新型酶抑制剂的努力。

DESCRIPTION (provided by applicant): The hallmark of enzyme catalysis is the high affinity of enzymes for their transition states, but the origin of this tight binding is generally not well understood. This application describes experiments to probe the mechanism by which the large intrinsic binding energy of substrate phosphodianion groups is utilized by a variety of enzymes to stabilize the transition state for formation of an unstable carbanion intermediate. Our central hypothesis is that flexible "phosphate gripper" loops are wide-spread conserved protein elements that provide binding energy that may be utilized for stabilization of the transition state for formation of an enzyme-bound carbanion. The enzymatic reactions of interest include aldose-ketose isomerization, sugar epimerization, aldol condensation and decarboxylation. We will use our "two-part substrate" protocol, where the substrate phosphodianion group, modeled by exogenous phosphite dianion, is detached from the portion of the substrate that undergoes chemical reaction. The specific transition state stabilization from phosphodianion binding interactions can then be quantified from the observed activation of the enzyme by exogenous phosphite dianion towards catalysis of the reaction of the second substrate fragment. Four projects are described to examine both the generality and the mechanism of transition state stabilization arising from flexible loop-phosphite interactions. (1) We will examine members of the orotidine 5'-monophosphate decarboxylase (OMPDC) superfamily. The goal is to determine whether the structurally conserved phosphate gripper loops of these enzymes share the common function of providing specific stabilization of carbanion intermediates. (2) We will examine the relationship between the length of the phosphate gripper loop and the utilization of enzyme-phosphodianion binding interactions for two enzymes that catalyze epimerization of phosphorylated sugars differing in length by only one carbon atom. (3) We will probe the mechanism by which OMPDC achieves its enormous 1017-fold rate acceleration for the chemically difficult decarboxylation of orotidine 5'-monophosphate. We will probe the mechanism by which interactions between the enzyme and the substrate phosphodianion are utilized in stabilization of the transition state for decarboxylation at the distant pyrimidine ring, and address other questions about the enigmatic mechanism of action of this enzyme. (4) We will continue our studies of the role of flexible loop-phosphodianion interactions in stabilization of the transition state for formation of the enediol(ate) intermediate of the aldose-ketose isomerization of triose phosphates catalyzed by triose phosphate isomerase. A major goal is to provide a full description of the physical mechanism by which the movement of flexible catalytic loops acts as a "switch" to turn on stabilizing transition state interactions. PUBLIC HEALTH RELEVANCE: Enzyme catalysts are one of the principal components of all living systems, and there are many diseases that arise from the malfunction or deficiency of only a single enzyme. Advances in the understanding of enzyme catalysis from mechanistic studies of enzymes and of nonenzymatic reactions may prove critical for drug design, to the understanding of metabolic pathways and diseases, and to the resolution of other health-related issues. The focus of this application is the critical role of flexible phosphate gripper loops in enzymatic catalysis and the results may spur efforts to develop novel enzyme inhibitors that specifically target these loops.