Reaction Specificity of Pyridoxal Phosphate Enzymes

磷酸吡哆醛酶的反应特异性

• 批准号: 7418818
• 负责人: Michael Toney
• 金额: $ 30.55万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2008-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7418818
• 关键词: 4-Aminobutyrate aminotransferase; Active Sites; Address; Alanine; Alanine Racemase; Amines; Amino Acids; Anabolism; Apoenzymes; Aspartate; Aspartate Transaminase; Binding; Carboxy-Lyases; Cell Wall; Coenzymes; Comparative Study; Complex; Decarboxylation; Dependence; Diaminopimelate decarboxylase; Enzymes; Free Energy; Grant; Measures; Metabolism; Modeling; Mutation; Nitrogen; Numbers; Ornithine Decarboxylase; Pyridoxal Phosphate; Pyridoxamine; Pyruvate; Pyruvates; Racemases; Reaction; Specificity; Statistical Methods; Structure; Temperature; Testing; aspartate 4-decarboxylase; design; enzyme activity; inhibitor/antagonist; mutant; nitrogen metabolism; phenylalanine (histidine) aminotransferase; protonation; racemization; research study; transamination

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. Active site mutants of DGD will be characterized and inhibitors synthesized and the structures of their complexes solved to determine how reaction specificity is enforced. Alanine racemase is the prototypical PLP dependent racemase, which provides D-alanine for bacterial cell wall biosynthesis. Studies with active site mutants will be pursued to understand how racemization specificity is tightly enforced. Additionally, free energy profile determination from global analysis of progress curves will be extended to include the temperature dependence of a mesophilic and thermophilic alanine racemase, and statistical methods will be developed that will allow model testing using global analysis. Free energy profiles will also be determined as a function of the fundamental extrinsic variables (e.g. pH, salt, temperature) controlling enzyme activity. The determination of isotopic free energy profiles will be completed, 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 serial transaminationdecarboxylation- transamination steps are controlled by the enzyme compared to pure transamination by aspartate aminotransferase. 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 ElEs on external aldimine formation, and by measuring C-H pKa's of enzyme-bound substrates.

磷酸吡哆醛（PLP）依赖性酶在氮代谢中普遍存在，并且催化许多 医学上重要的转变作为一个群体，它们催化了非常广泛的反应。一 与抑制剂设计直接相关的基本问题是给定的脱辅基酶如何决定一个独特的 反应特异性二烷基甘氨酸脱羧酶（DGD）是一种不常见的PLP依赖性酶， 在其正常的催化循环中催化脱羧和转氨作用。这使得详细的 探索立体电子效应，这是确定PLP反应的主要机制 的特异性DGD的活性位点突变体将被表征，抑制剂将被合成， 它们的复合物被解析以确定反应特异性是如何被加强的。丙氨酸消旋酶是 原型PLP依赖性消旋酶，其为细菌细胞壁生物合成提供D-丙氨酸。研究 与活性位点突变体将被追求，以了解如何外消旋特异性是严格执行。 此外，从进度曲线的全局分析确定自由能分布将扩展到 包括嗜温和嗜热丙氨酸消旋酶的温度依赖性，以及统计学 将制定方法，允许使用全球分析进行模型测试。自由能源配置文件也将 作为基本外在变量（例如pH、盐、温度）控制的函数来确定 酶活性将完成同位素自由能分布的测定，提供下列影响： 所有基本步骤上都是氘化。二氨基庚二酸反应特异性的比较研究 在最后一次授予期间启动的脱羧酶和鸟氨酸脱羧酶将扩大到 确定这些同源酶之间反应特异性差异的起源。一个新项目 天冬氨酸β-脱羧酶将启动了解如何串行transaminationdecarboxylase- 与纯转氨作用相比，转氨作用步骤由酶控制， 天门冬氨酸氨基转移酶。最后，PLP酶的亲电要求将通过以下方法确定： 15 N NMR实验，其中PLP酶的活性位点氮的质子化状态将是 用辅酶类似物与吡哆胺丙酮酸转氨酶测定， 外部醛亚胺的形成，并通过测量C-H pKa的酶结合的底物。