Coordination of functions by proline metabolic proteins

描述（由申请人提供）：代谢是由酶的复杂网络组成的，这些酶通过共享底物和产物分子形成连接。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，或者从一种酶到另一种酶。一般来说，活性分子在酶活性位点之间传递的机制尚不清楚。这个项目的目标是了解脯氨酸分解代谢的中间体是如何从脯氨酸脱氢酶（PRODH）转移到？1-吡咯-5-羧酸脱氢酶（P5CDH）。PRODH是一种黄酶，它催化脯氨酸氧化为P5C。P5C是一种反应性分子，与反应性羰基类谷氨酸半醛（GSA）形成非酶促、ph依赖的平衡。P5CDH是一种NAD+依赖酶，催化GSA氧化为谷氨酸。中间体P5C/GSA在脯氨酸分解代谢和合成途径以及精氨酸生物合成途径中都是常见的。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。本项目将以细菌双功能酶脯氨酸利用酶（Proline utilization A, PutA）为模型，详细了解通道。在PutAs中，PRODH和P5CDH融合成一个单一的大蛋白。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，假设它既是P5C水解成GSA的反应室，也是促进GSA运输到P5CDH活性位点的保护途径。本文报道的稳态和快速反应动力学数据也支持PutA的通道机制。这些初步的观察结果进一步提出了单功能的PRODH和P5CDH酶，如在人类中发现的，相互作用并参与分子间通道的假设。人类酶的细菌同源物中的通道也将被研究。这一建议有三个目的：1。建立puta衬底通道的结构和动力学框架。2. 研究PutA衬底沟槽形成的机理。3. 探索单功能PRODH和P5CDH中的底物通道和蛋白质相互作用。完成这些目标将提供一个全面而详细的了解脯氨酸分解代谢中的底物通道。公共卫生相关性：该项目提出了详细的生化和结构研究酶的氨基酸脯氨酸的循环氧化为谷氨酸。这些酶的遗传缺陷导致高脯氨酸血症紊乱，这可能与智力迟钝、高热性癫痫发作的频率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，通过在肿瘤抑制因子p53介导的细胞死亡级联反应中作为活性氧发生器，有助于减少人类的致癌作用。拟议的研究将研究如何在一个被称为底物通道的过程中，在脯氨酸再循环酶之间传递活性中间体。有人提出，通道是脯氨酸氧化过程的一个基本方面。

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.