Coordination of functions by proline metabolic proteins

脯氨酸代谢蛋白的功能协调

• 批准号: 7527359
• 负责人: JOHN J TANNER
• 金额: $ 28.65万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-05 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7527359
• 关键词: Active Sites; Address; Amino Acids; Anabolism; Apoptosis; Arginine; Bacteria; Biochemical; Biological Process; Bradyrhizobium; Brain Diseases; Catabolism; Cell Death; Cellular Stress Response; Communication; Complex; Crystallography; Data; Data Reporting; Diffuse; Disease; Distal; Energy Metabolism; Environment; Enzymes; Equilibrium; Eukaryota; Eukaryotic Cell; Febrile Convulsions; Frequencies; Generations; Glutamates; Goals; Homologous Gene; Human; Hydrolysis; Inborn Genetic Diseases; Kinetics; Label; Lead; Link; Malignant Neoplasms; Mediating; Mental Retardation; Metabolic; Metabolism; Methods; Modeling; Motion; Movement; Mutation; Oxidation-Reduction; Oxidoreductase; Pathway interactions; Predisposition; Process; Proline; Proline Dehydrogenase; Protein p53; Proteins; Public Health; Puncture procedure; RNA; Reaction; Reactive Oxygen Species; Recycling; Research; Resolution; Scheme; Schizophrenia; Site-Directed Mutagenesis; Solvents; Structure; System; Testing; Translation Initiation; Two-Hybrid System Techniques; Work; carcinogenesis; delta-1-pyrroline-5-carboxylate; in vivo; mutant; oxidation; protein protein interaction

DESCRIPTION (provided by applicant): Metabolism is built of complex networks of enzymes that form links by sharing substrate and product molecules. Some of these shared molecules, which are often reactive, are not freely diffusing, but rather, their motion is directed, or channeled from one enzyme to another. In general, the mechanisms by which reactive molecules are passed between enzyme active sites are poorly understood. The goal of this project is to understand how the intermediates of proline catabolism are channeled from proline dehydrogenase (PRODH) to ?1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH). PRODH is a flavoenzyme that catalyzes the oxidation of proline to P5C. P5C is a reactive molecule that forms a nonenzymatic, pH-dependent equilibrium with the reactive carbonyl species glutamic semialdehyde (GSA). P5CDH is an NAD+dependent enzyme that catalyzes the oxidation of GSA to glutamate. The intermediate P5C/GSA is common to both the proline catabolic and synthetic pathways, and to arginine biosynthesis. P5C/GSA also influences many biological processes, including apoptosis, reactive oxygen species generation and RNA translation initiation. This project will use the bacterial bifunctional enzyme, Proline utilization A (PutA), as a model to understand channeling in detail. In PutAs, PRODH and P5CDH are fused into a single, large protein. The recent crystal structure of a PutA has revealed a unique system of internal cavities and tunnels that is hypothesized to function as both a reaction chamber for the hydrolysis of P5C to GSA and a protected pathway that facilitates transport of GSA to the P5CDH active site. Steady-state and rapid reaction kinetic data reported here also support a channeling mechanism for PutA. These initial observations furthermore suggest the hypothesis that monofunctional PRODH and P5CDH enzymes, such as those found in humans, interact and engage in intermolecular channeling. Channeling in bacterial homologs of the human enzymes will also be studied. This proposal has three aims: 1. Establish the structural and kinetic framework underlying substrate channeling in PutAs. 2. Investigate the mechanism of substrate channeling in PutA. 3. Explore substrate channeling and protein-protein interactions in monofunctional PRODH and P5CDH. Completion of these aims will provide a comprehensive, yet detailed, understanding of substrate channeling in proline catabolism. PUBLIC HEALTH RELEVANCE: This project proposes detailed biochemical and structural studies of the enzymes that recycle the amino acid proline by oxidizing it to glutamate. Genetic defects in these enzymes lead to hyperprolinemia disorders, which can be associated with mental retardation, higher frequency of febrile seizures and increased susceptibility to the disabling brain disorder schizophrenia. Also, one of the enzymes, proline dehydrogenase, helps reduce carcinogenesis in humans by serving as a reactive oxygen species generator in the cell death cascade mediated by tumor suppressor p53. The proposed research will examine how reactive intermediates are passed between proline recycling enzymes in a process known as substrate channeling. It is proposed that channeling is a fundamental aspect of the proline oxidation process.

描述（由申请人提供）：代谢是由复杂的酶网络构建的，这些酶通过共享底物和产物分子形成链接。这些共享分子中的一些通常是反应性的，不是自由扩散的，而是它们的运动是定向的，或者从一种酶引导到另一种酶。一般来说，人们对反应分子在酶活性位点之间传递的机制知之甚少。该项目的目标是了解脯氨酸分解代谢的中间体如何从脯氨酸脱氢酶（PRODH）引导至β1-吡咯啉-5-羧酸（P5C）脱氢酶（P5CDH）。 PRODH 是一种黄素酶，可催化脯氨酸氧化为 P5C。 P5C 是一种反应性分子，可与反应性羰基谷氨酸半醛 (GSA) 形成非酶促、pH 依赖性平衡。 P5CDH 是一种 NAD+ 依赖性酶，可催化 GSA 氧化为谷氨酸。中间体 P5C/GSA 对于脯氨酸分解代谢和合成途径以及精氨酸生物合成都是常见的。 P5C/GSA 还影响许多生物过程，包括细胞凋亡、活性氧生成和 RNA 翻译起始。该项目将使用细菌双功能酶脯氨酸利用 A (PutA) 作为模型来详细了解通道。在 PutAs 中，PRODH 和 P5CDH 融合成单个大蛋白质。最近的 PutA 晶体结构揭示了一个独特的内部空腔和隧道系统，假设该系统既可作为 P5C 水解为 GSA 的反应室，又可作为促进 GSA 运输至 P5CDH 活性位点的受保护途径。这里报告的稳态和快速反应动力学数据也支持 PutA 的通道机制。这些初步观察结果还提出了这样的假设：单功能 PRODH 和 P5CDH 酶（例如在人类中发现的酶）相互作用并参与分子间通道。人类酶的细菌同系物的通道也将被研究。该提案具有三个目标： 1. 建立 PutAs 中底物通道的结构和动力学框架。 2. 研究PutA中底物沟道的机制。 3. 探索单功能 PRODH 和 P5CDH 中的底物通道和蛋白质-蛋白质相互作用。完成这些目标将为脯氨酸分解代谢中的底物通道提供全面而详细的理解。公共健康相关性：该项目提出了对通过将氨基酸脯氨酸氧化为谷氨酸来回收氨基酸的酶进行详细的生化和结构研究。这些酶的遗传缺陷会导致高脯氨酸血症，这可能与智力低下、热性惊厥频率升高以及对致残性脑部疾病精神分裂症的易感性增加有关。此外，其中一种酶脯氨酸脱氢酶在肿瘤抑制因子 p53 介导的细胞死亡级联中充当活性氧发生器，有助于减少人类的致癌作用。拟议的研究将研究反应中间体如何在称为底物通道的过程中在脯氨酸回收酶之间传递。有人提出沟道是脯氨酸氧化过程的一个基本方面。