Coordination of functions by proline metabolic proteins

描述(申请人提供)：新陈代谢是由复杂的酶网络构建的，这些网络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部位之间传递的机制知之甚少。本项目的目的是了解脯氨酸分解代谢的中间产物是如何从脯氨酸脱氢酶(PROH)传导到1-吡咯烷-5-羧酸(P5C)脱氢酶(P5CDH)的。PROSH是一种黄素酶，催化脯氨酸氧化为P5C。P5C是一种反应性分子，它与反应性羰基物种谷氨酸半醛(GSA)形成非酶、pH依赖的平衡。P5CDH是一种NAD依赖的酶，催化GSA氧化成谷氨酸。中间产物P5C/GSA在脯氨酸分解代谢途径和合成途径以及精氨酸生物合成途径中都是共有的。P5C/GSA还影响许多生物学过程，包括细胞凋亡、活性氧的产生和RNA翻译的启动。这个项目将使用细菌的双功能酶，脯氨酸利用A(PUTA)作为一个模型来详细了解通道作用。在Putas中，PROSH和P5CDH被融合成一个单一的大蛋白。最近PUTA的晶体结构揭示了一个独特的内部空腔和隧道系统，该系统被认为既是P5C水解为GSA的反应室，也是促进GSA运输到P5CDH活性部位的受保护途径。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。这些初步观察进一步表明，单功能的PROH和P5CDH酶，如那些在人类中发现的酶，相互作用并参与分子间通道。人类酶的细菌同源物中的通道也将被研究。这一建议有三个目标：1.建立普塔斯底物通道的结构和动力学框架。2.研究了PUTA中基质沟道的形成机制。3.探讨单功能Proth和P5CDH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，理解底物在脯氨酸分解代谢中的通道。与公众健康相关：该项目建议对通过将氨基酸Pro氧化为谷氨酸来循环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、热性癫痫的更高频率以及对致残性大脑障碍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，通过在肿瘤抑制基因p53介导的细胞死亡级联反应中充当活性氧物种生成器，帮助减少人类的癌症发生。这项拟议的研究将研究活性中间体如何在被称为底物通道的过程中在脯氨酸循环酶之间传递。有人提出，沟道作用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.