ENZYMES AND INHIBITORS OF L-PIPECOLIC ACID METABOLISM

L-哌啶酸代谢的酶和抑制剂

• 批准号: 2460570
• 负责人: T. MARK ZABRISKIE
• 金额: $ 7.57万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2460570
• 关键词: GABA receptor; Macaca mulatta; aminoacid analog; aminoacid inhibitor; aminoacid metabolism; aminoacid oxidase; brain metabolism; cyclic aminoacid; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; laboratory rabbit; lysine; mitochondria; proline

The overall goals of the proposed research project are to address fundamental questions related to the enzymology of L-pipecolic acid metabolism in the mammalian brain and to develop potent, specific inhibitors of its degradation. Such inhibitors represent a potential new means for developing anticonvulsive agents and for studying GABA receptor complexes. L-Pipecolic (L-PA) acid is a six-carbon cyclic amino acid, the higher homologue of L-proline, and is a minor product of lysine metabolism in various organisms and most mammalian tissues. The notable exception is the brain, where lysine is primarily degraded to L-PA which has been shown to possess both neuromodulating and anticonvulsive properties. A large body of evidence supports an interaction of L-pipecolate and GABAergic transmission, either at GABA receptors or through binding to its own receptor and exerting an allosteric effect on the GABA complex. Hence, a primary objective of this proposal is to define the features of the key enzyme involved in L-pipecolic acid catabolism in the brain and use this information to develop specific inactivators of it, thereby decreasing L- PA degradation and elevating its concentration and neurological effects. In liver and kidney, the first step of L-PA degradation is known to be species dependent, and is catalyzed by either a flavin-containing peroxisomal oxidase or mitochondrial dehydrogenase. Rhesus monkey liver L- pipecolate oxidase has been purified, but nothing is known about the enzymology of L-PA metabolism in the brain or about the traits of the mitochondrial enzyme from any source. Thus, the first specific goals of this project will be to isolate and fully characterize mitochondrial L- pipecolate oxidizing enzymes from rabbit kidney and brain, and the peroxisomal enzyme from primate brain. This will generate data leading to a better understanding of L-PA metabolism in general and presents a unique opportunity to study different enzymes catalyzing the same reaction in different mammalian species. Complete analysis of these enzymes will include: determining physical and kinetic properties; detailed cofactor studies; and stereochemical and mechanistic relatedness of the enzymes isolated from the different species and organelles. The second specific aim of this project involves designing and constructing modified substrates and potent inhibitors of L-PA oxidation. The focus will be on mechanism-based inactivators designed to exploit features characteristic of flavin-dependent amine oxidases. These inactivators will serve as valuable probes of the mechanisms and active site environments of the various L-pipecolate oxidizing enzymes. Inhibitors designed to develop highly reactive radical or electrophilic species could form covalent adducts with the target enzymes and permit active site sequencing and comparisons. Potent inactivators may unveil a new route to designing therapeutically useful anticonvulsants and the pipecolate analogs developed in this work may serve as valuable pharmacological ligands for studying and classifying GABA receptors.

拟议研究项目的总体目标是解决 与L-哌啶酸酶学有关的基本问题 哺乳动物大脑中的代谢，并开发有效的，特异性的 抑制其降解。这种抑制剂代表了一种潜在的新的 用于开发抗惊厥剂和研究GABA受体的方法 配合物L-哌啶酸（L-PA）是一种六碳环氨基酸， L-脯氨酸的高级同系物，是赖氨酸代谢的次要产物 存在于各种生物和大多数哺乳动物组织中。值得注意的例外是 在大脑中，赖氨酸主要降解为L-PA， 具有神经调节和抗惊厥的特性。大 大量证据支持L-哌啶酸盐和GABA能的相互作用 传递，无论是在GABA受体或通过结合到自己的 受体并对GABA复合物产生变构作用。于是，一 本提案的主要目标是定义密钥的特征 酶参与的L-哌啶酸催化剂在大脑中，并使用这种 信息开发特定的灭活剂，从而减少L- PA降解和提高其浓度和神经效应。 在肝脏和肾脏中，已知L-PA降解的第一步是 物种依赖性，并由含有黄素的 过氧化物酶体氧化酶或线粒体脱氢酶。恒河猴肝L- 哌啶酸氧化酶已被纯化，但没有什么是已知的， L-PA在脑中代谢的酶学或关于L-PA代谢的特征， 任何来源的线粒体酶。因此，第一个具体目标 该项目将分离和充分表征线粒体L- 从兔肾和脑中提取哌啶酸氧化酶， 灵长类动物大脑中的过氧化物酶体。这将产生数据， 更好地了解L-PA代谢的一般，并提出了一个独特的 有机会研究不同的酶催化相同的反应， 不同的哺乳动物物种。对这些酶的完整分析将 包括：确定物理和动力学性质;详细辅因子 研究;以及酶的立体化学和机械相关性 分离自不同的物种和细胞器。 该项目的第二个具体目标是设计和 构建修饰的底物和L-PA氧化的有效抑制剂。 重点将放在基于机制的灭活剂上， 具有黄素依赖性胺氧化酶的特征。这些 灭活剂将作为机制和活性的有价值的探针 各种L-哌啶酸氧化酶的位点环境。 设计用于产生高活性自由基或亲电子基团的抑制剂 物种可以与靶酶形成共价加合物， 活性位点测序和比较。有效的灭活剂可能会揭示一种 设计治疗上有用的抗惊厥药物的新途径， 在这项工作中开发的哌啶类似物可能是有价值的 用于研究和分类GABA受体的药理学配体。