Reaction Specificity of Pyridoxal Phosphate Enzymes

描述（由申请人提供）：磷酸吡哆醛（PLP）依赖性酶在氮代谢中普遍存在，并催化许多医学上重要的转化。作为一个群体，它们催化了非常广泛的反应。抑制剂设计的一个基本问题是给定的脱辅基酶如何决定独特的反应特异性。二烷基甘氨酸脱羧酶（DGD）是一种不寻常的PLP依赖性酶，在其正常的催化循环中快速催化脱羧和转氨。这允许立体电子效应的定量，这是用于确定PLP反应特异性的主要机制。氧化脱羧特异性在DGD中通过协同脱羧/质子转移过渡态实现。这个协调过渡态的能量需求将被确定。此外，DGD具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。了解实现对碱金属离子的特异性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，其为细菌细胞壁生物合成提供D-丙氨酸。这种酶在去质子化碳时非常快（约20，000 s [-1]），并显示出非常逼真的保真度。据推测，这些是一个协调的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谱将通过直接动力学分析作为控制酶活性的基本外在变量（例如pH、盐、温度）的函数来确定，从而提供对酶促反应中能量屏障的起源的洞察。人丝氨酸消旋酶在脑中提供D-丝氨酸，其是NMDA受体的共活化剂。这种酶将与丙氨酸消旋酶进行机械比较，并作为预防中风损伤的药物的靶点进行检查。最后，PLP酶的亲电要求将通过协作15N NMR研究来确定，其中确定PLP酶的活性位点硝化剂的质子化状态，并且另外通过使用电子等排辅酶类似物来确定。

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.