Coordination of functions by proline metabolic proteins

脯氨酸代谢蛋白的功能协调

• 批准号: 7653787
• 负责人: JOHN J TANNER
• 金额: $ 27.39万
• 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/7653787
• 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; 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; 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; carboxylate; carcinogenesis; in vivo; mutant; oxidation; protein protein interaction; public health relevance; pyrroline; 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; 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; 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; carboxylate; carcinogenesis; in vivo; mutant; oxidation; protein protein interaction; public health relevance; pyrroline

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品分子形成链接。这些共享分子中的一些通常是反应性的，不是自由扩散的，而是将其运动引向或从一种酶引导到另一种酶。通常，在酶活性位点之间通过反应性分子传递的机制知之甚少。该项目的目的是了解如何将脯氨酸分解代谢的中间体从脯氨酸脱氢酶（PODH）传递到？1-吡咯氨酸-5-羧酸盐（P5C）脱氢酶（P5CDH）。 PODH是一种黄酮酶，可催化脯氨酸的氧化为P5C。 P5C是一种反应性分子，与反应性羰基物种谷氨酰胺半二醛（GSA）形成非酶的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P5C/GSA是脯氨酸分解代谢和合成途径以及精氨酸生物合成的共同点。 P5C/GSA还影响许多生物学过程，包括凋亡，活性氧的产生和RNA转化启动。该项目将使用细菌双功能酶，脯氨酸利用A（PUTA）作为详细了解通道的模型。在Puta中，PODH和P5CDH融合成单个大蛋白质。 PUTA的最新晶体结构揭示了一种独特的内部空腔和隧道系统，该系统被认为是P5C水解为GSA水解的反应室，又是一个受保护的途径，可促进GSA向P5CDH活性位点运输。这里报告的稳态和快速反应动力学数据还支持PUTA的通道机制。这些最初的观察结果进一步表明了一个假设，即单官能的PODH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。还将研究在人类酶的细菌同源物中的介导。该提议具有三个目标：1。建立PUTAS基板渠道基础的结构和动力学框架。 2。研究PUTA中底物通道的机理。 3。探索单功能PODH和P5CDH中的底物通道和蛋白质 - 蛋白质相互作用。这些目标的完成将提供对脯氨酸分解代谢中底物渠道的全面而详细的理解。公共卫生相关性：该项目提出了对酶的详细生化和结构研究，这些酶通过将其氧化为谷氨酸来回收氨基酸脯氨酸。这些酶中的遗传缺陷会导致高蛋白血症疾病，这可能与智力低下，更高的发热性癫痫发作频率以及对残疾脑疾病精神分裂症的敏感性增加。此外，其中一种酶是脯氨酸脱氢酶，通过用作由肿瘤抑制p53介导的细胞死亡级联反应性氧气生成器，有助于减少人类的癌变。拟议的研究将研究在称为底物通道的过程中脯氨酸回收酶之间的反应性中间体。有人提出，通道是脯氨酸氧化过程的基本方面。