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。直接影响抑制剂设计的一个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。现在我们将分析DGD的关键活性位点残基的机理。丙氨酸消旋酶（AlaR）是典型的PLP依赖性消旋酶，为细菌细胞壁的生物合成提供d -丙氨酸。从进程曲线的全局分析中确定的自由能剖面将与AlaR一起扩展，以包括嗜中温和嗜热AlaR的温度依赖性，并且将开发统计方法，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。将扩展对同位素自由能剖面的测定，提供氘对所有基本步骤的影响。将扩大对上一个授予期启动的二氨基苯甲酸脱羧酶和鸟氨酸脱羧酶的反应特异性的比较研究，以确定这些同源酶之间反应特异性差异的来源。一个关于天冬氨酸β -脱羧酶的新项目将启动，以了解该酶在复杂的反应序列中如何控制反应特异性。最后，PLP酶的亲电性要求将通过15N NMR实验来确定，其中PLP酶的活性位点氮的质子化状态将通过使用与吡哆胺丙酮酸转氨酶的辅酶类似物，通过测定外部醛胺形成的EIEs和测量酶结合底物的C-H pKa来确定。公共卫生相关性：利用维生素B6使代谢反应更快的医学上重要的酶将在机制细节上进行研究，以了解这大类酶如何控制由哪种底物制成哪种产品（即这些酶如何控制反应特异性）。几种维生素B6依赖酶是目前使用的药物的靶点，我们将研究的几种酶是极好的药物靶点。我们的研究将为小分子药物高度特异性地靶向这些酶提供所需的基础知识。

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.