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。一个直接关系到抑制剂设计的基本问题是，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。这允许详细探索立体电子效应，这是确定PLP反应特异性的主要机制。我们现在将分析DGD的机械临界活性中心残留物。丙氨酸消旋酶(ALAR)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。从进展曲线的全局分析中确定的自由能分布将扩展到ALAR，以包括中温和嗜热ALAR的温度依赖关系，并将开发统计方法，允许使用全局分析进行模型测试。还将确定几个活性位点突变体的自由能分布。同位素自由能分布的测定将被扩大，提供所有基本步骤上的氚效应。在最后一次授权期启动的二氨基松酸脱羧酶和鸟氨酸脱羧酶的反应特异性的比较研究将被扩大，以确定这些同源酶反应特异性差异的来源。将启动一个关于天冬氨酸β-脱羧酶的新项目，以了解该酶所采用的复杂反应序列中反应特异性是如何控制的。最后，将通过15N核磁共振实验确定PLP酶的亲电性要求，其中将通过使用吡哆胺丙酮酸氨基转移酶的辅酶类似物，通过测定外源乙二胺形成的EES和通过测量酶结合底物的C-H pKA来确定PLP酶活性部位氮质子化状态。公共卫生相关性：将详细研究利用维生素B6使代谢反应更快的医学上重要的酶，以了解这一大类酶如何控制哪种底物生产哪种产品(即这些酶如何控制反应特异性)。几种依赖维生素B6的酶是目前使用的药物靶点，我们将研究的几种酶是很好的药物靶点。我们的研究将提供高度靶向这些酶所需的基础知识，特别是小分子药物。

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 directly 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 a detailed exploration of stereoelectronic effects, which are a primary mechanism for determining PLP reaction specificity. We will now analyze mechanistically critical active site residues of DGD. Alanine racemase (AlaR) is the prototypical PLP dependent racemase, which provides D-alanine for bacterial cell wall biosynthesis. Free energy profile determination from global analysis of progress curves will be extended with AlaR to include the temperature dependence of a mesophilic and thermophilic AlaR, and statistical methods will be developed that will allow model testing using global analysis. Free energy profiles will also be determined for several active site mutants. The determination of isotopic free energy profiles will be extended, providing the effects of deuteration on all elementary steps. Comparative studies on the reaction specificity of diaminopimelate decarboxylase and ornithine decarboxylase initiated during the last granting period will be expanded to determine the origins of reaction specificity differences between these homologous enzymes. A new project on aspartate beta-decarboxylase will be initiated to understand how the reaction specificity is controlled in the complex reaction sequence employed by this enzyme. Lastly, the electrophilic requirements of PLP enzymes will be determined with 15N NMR experiments in which the protonation state of active site nitrogens of PLP enzymes will be determined, by using coenzyme analogs with pyridoxamine pyruvate aminotransferase, by determinining EIEs on external aldimine formation and by measuring C-H pKa's of enzyme-bound substrates. PUBLIC HEALTH RELEVANCE: Medically important enzymes that utilize vitamin B6 to make metabolic reactions go faster will be studied in mechanistic detail to understand how this large class of enzymes controls which product is made from which substrate (i.e. how these enzymes control reaction specificity). Several vitamin B6 dependent enzymes are targets of currently employed pharmaceuticals, and several of the enzymes we will study are excellent drug targets. Our studies will provide the fundamental knowledge required to target these enzymes highly specifically with small molecule drugs.