Reaction Specificity of Pyridoxal Phosphate Enzymes

描述（由申请人提供）：磷酸吡哆醛（PLP）依赖酶在氮代谢中普遍存在，并催化许多重要的医学转化。作为一个群体，它们能催化各种各样的反应。一个与抑制剂设计有关的基本问题是给定的脱酶如何决定独特的反应特异性。二烷基甘氨酸脱羧酶（DGD）是一种罕见的PLP依赖性酶，在其正常的催化循环中快速催化脱羧和转氨化。这允许立体电子效应的定量，这是确定PLP反应特异性的主要机制。氧化脱羧特异性是通过协同脱羧/质子转移过渡态在DGD中实现的。这种协调过渡状态的能量需求将被确定。此外，DGD有两个碱金属离子结合位点，其中一个结合多种离子并控制活性。了解碱金属离子特异性产生的机制具有广泛的生理意义。丙氨酸消旋酶是典型的PLP依赖性消旋酶，它为细菌细胞壁的生物合成提供d -丙氨酸。这种酶使碳去质子化的速度非常快（大约20000秒[-1]），并且表现出非凡的保真度。据推测，这是一个协调的双质子转移过渡态的结果。这一假设将得到验证。此外，丙氨酸消旋酶的自由能谱将通过直接的动力学分析确定为控制酶活性的基本外部变量（如pH，盐，温度）的函数，从而深入了解酶反应中能量垒的起源。人类丝氨酸消旋酶在大脑中提供d -丝氨酸，这是NMDA受体的共激活剂。该酶将与丙氨酸消旋酶进行机理比较，并作为预防中风损伤的药物靶点进行研究。最后，PLP酶的亲电性要求将通过15N核磁共振研究确定，其中确定了PLP酶活性位点氮的质子化状态，另外还使用了等同辅酶类似物。

DESCRIPTION (provided by applicant): Pyridoxal phosphate (PLP) dependent enzymes are ubiquitous in nitrogen metabolism and catalyze many medically important transformations. As a group, they catalyze an extraordinarily wide variety of reactions. A fundamental question bearing on inhibitor design is how a given apoenzyme determines a unique reaction specificity. Dialkylglycine decarboxylase (DGD) is an unusual PLP dependent enzyme that rapidly catalyzes both decarboxylation and transamination in its normal catalytic cycle. This allows the quantitation of stereoelectronic effects, which are a primary mechanism for determining PLP reaction specificity. Oxidative decarboxylation specificity is achieved in DGD via a concerted decarboxylation/proton transfer transition state. The energetic requirements of this concerted transition state will be determined. Additionally, DGD has two alkali metal ion binding sites, one of which binds a variety of ions and controls activity. Understanding the mechanism by which specificity for alkali metal ions is achieved has broad physiological significance. Alanine racemase is the prototypical PLP dependent racemase, which provides D-alanine for bacterial cell wall biosynthesis. This enzyme is extremely fast (about 20,000 s[-1]) at deprotonating carbon and shows extraordinarily fidelity. It is hypothesized that these are a result of a concerted double proton transfer transition state. This hypothesis will be tested. Additionally, the free energy profile for alanine racemase will be determined by straightforward kinetic analyses as a function of the fundamental extrinsic variables (e.g. pH, salt, temperature) controlling enzyme activity, providing insight into the origins of energy barriers in enzymatic reactions. Human serine racemase provides D-serine in the brain, which is a coactivator of the NMDA receptor. This enzyme will be compared mechanistically with alanine racemase and examined as a target for drugs to prevent stroke damage. Lastly, the electrophilic requirements of PLP enzymes will be determined through a collaborative 15N NMR study in which the protonation state of active site nitzogens of PLP enzymes is determined, and additionally by using isosteric coenzyme analogs.