Reaction Specificity of Pyridoxal Phosphate Enzymes

描述(申请人提供)：磷酸吡哆醛(PLP)依赖的酶在氮代谢中普遍存在，并催化许多医学上重要的转化。作为一个基团，它们催化了非常广泛的各种反应。与抑制剂设计有关的一个基本问题是，给定的脱辅酶酶如何确定唯一的反应特异性。二烷基甘氨酸脱羧酶(DGD)是一种不常见的PLP依赖酶，在其正常的催化循环中能快速催化脱羧基和转氨基。这允许对立体电子效应进行量化，这是确定PLP反应特异性的主要机制。在DGD中，氧化脱羧基的特异性是通过协调的脱羧基/质子转移过渡态实现的。这种协调一致的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部位，其中一个结合多种离子并控制活性。了解碱金属离子获得特异性的机制具有广泛的生理学意义。丙氨酸消旋酶是典型的依赖PLP的消旋酶，为细菌细胞壁的生物合成提供D-丙氨酸。这种酶去质子化碳的速度极快(约20,000 S[-1])，并表现出非凡的保真度。假设这是一个协调双质子转移过渡态的结果。这一假设将得到检验。此外，丙氨酸消旋酶的自由能分布将通过直接的动力学分析作为控制酶活性的基本外部变量(如pH、盐、温度)的函数来确定，从而深入了解酶反应中能垒的起源。人类丝氨酸消旋酶在大脑中提供D-丝氨酸，它是NMDA受体的辅助激活剂。这种酶将从机理上与丙氨酸消旋酶进行比较，并作为预防中风损害的药物的靶点进行研究。最后，PLP酶的亲电性需求将通过合作的15N核磁共振研究来确定，在该研究中，PLP酶的活性部位亚硝酸质子化状态被确定，此外，还将通过使用异构化辅酶类似物来确定。

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.