ENZYMES AND INHIBITORS OF L-PIPECOLIC ACID METABOLISM

拟议研究项目的总体目标是解决 L的酶学基础问题--吡喃甲酸 哺乳动物大脑中的新陈代谢，并发展出强大的、特异的 它的降解剂。这些抑制剂代表着一种潜在的新的 开发抗惊厥药物和研究GABA受体的方法 复合体。L-吡咯烷酸(L-PA)是一种六碳环状氨基酸， 是L-脯氨酸的高级同系物，是赖氨酸代谢的次要产物 在各种生物和大多数哺乳动物组织中。值得注意的例外是 大脑，赖氨酸在那里主要降解为L-PA，这已被证明 兼具神经调节和抗惊厥作用。大号 证据支持L-哌替酸和GABA能相互作用 传递，要么通过GABA受体，要么通过与自身结合 受体，并对GABA复合体施加变构效应。因此，一个 本提案的主要目标是定义关键字的特征 参与脑内L-吡啶甲酸分解代谢的酶及其应用 信息，以开发其特定的灭活剂，从而降低L- 苯丙氨酸的降解和浓度升高以及对神经的影响。 已知L-PA在肝脏和肾脏的第一步降解是 依赖于物种，并由含有黄素的 过氧化物酶或线粒体脱氢酶。恒河猴肝L 已经提纯了吡咯酸氧化酶，但对它的 L-PA在脑内代谢的酶学研究 任何来源的线粒体酶。因此，第一个具体目标是 本项目将分离并全面鉴定线粒体L-- 兔肾和脑中的哌甲酸氧化酶，以及 灵长类动物大脑中的过氧化物酶。这将生成数据，从而导致 更好地了解L-PA代谢的一般情况，并呈现独特的 研究催化同一反应的不同酶的机会 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；详细的辅因 研究；以及酶的立体化学和机械相关性 从不同的物种和细胞器中分离出来。 该项目的第二个具体目标涉及设计和 构建L-PA氧化的修饰底物和有效的抑制剂。 重点将放在基于机制的灭活剂上，旨在利用 黄素依赖的胺氧化酶的特征。这些 灭活剂将作为有价值的机制和活性探针 现场环境中的各种L-吡咯烷酸氧化酶。 设计用于开发高活性自由基或亲电性的抑制剂 物种可以与目标酶形成共价加合物，并允许 活性部位测序和比较。强大的灭活剂可能会揭开一种 设计治疗有用的抗惊厥药物的新途径和 在这项工作中开发的吡喃甲酸酯类似物可能是有价值的 用于研究和分类GABA受体的药理配体。

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.