Coordination of functions by proline metabolic proteins

描述（由申请方提供）：代谢由酶的复杂网络构成，这些酶通过共享底物和产物分子形成链接。这些共享的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，或从一种酶引导到另一种酶。一般来说，反应分子在酶活性位点之间传递的机制知之甚少。本项目的目标是了解脯氨酸催化剂的中间产物是如何从脯氨酸脱氢酶（PRODH）引导到？1-吡咯啉-5-羧酸（P5 C）脱氢酶（P5 CDH）。PRODH是催化脯氨酸氧化成P5 C的黄素酶。P5 C是一种活性分子，与活性羰基物质谷氨酸半醛（GSA）形成非酶促、pH依赖性平衡。P5 CDH是催化GSA氧化成谷氨酸的NAD+依赖性酶。中间体P5 C/GSA是脯氨酸分解代谢和合成途径以及精氨酸生物合成所共有的。P5 C/GSA还影响许多生物学过程，包括细胞凋亡、活性氧产生和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PutA中，PRODH和P5 CDH融合成单个大蛋白。最近的晶体结构的PutA揭示了一个独特的系统的内部空腔和隧道，假设作为一个反应室的水解P5 C GSA和保护途径，促进运输GSA的P5 CDH活性位点。稳态和快速反应动力学数据也支持PutA的通道机制。这些初步的观察进一步表明，单功能PRODH和P5 CDH酶，如在人类中发现的，相互作用，并参与分子间通道的假设。还将研究人类酶在细菌同系物中的降解。这项建议有三个目的：1。建立结构和动力学框架下的底物通道PutAs。2.研究了PutA中基底沟道的形成机理。3.探索单功能PRODH和P5 CDH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一个全面的，但详细的了解，在脯氨酸催化剂的底物通道。公共卫生相关性：该项目提出了详细的生物化学和结构的酶的研究，回收的氨基酸脯氨酸氧化成谷氨酸。这些酶的遗传缺陷会导致高脯氨酸血症，这可能与智力迟钝、高热惊厥频率较高以及对致残性脑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产生剂，有助于减少人类的致癌作用。拟议的研究将研究活性中间体是如何在脯氨酸回收酶之间传递的，这一过程被称为底物通道。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.