Coordination of functions by proline metabolic proteins.

 描述(申请人提供)：细胞中的代谢酶很少在孤立的情况下发挥作用。通常，它们的活动是通过彼此的物理联系和细胞结构来协调的。这些关联的结果是代谢中间产物不与细胞环境平衡，而是在酶之间传递。尽管人们普遍认识到蛋白质-蛋白质相互作用是普遍存在的，但底物通道的机制仍然相对较少被研究，因此知之甚少。我们通过探索底物在脯氨酸分解代谢的酶内和酶之间的通道来帮助缩小这一知识鸿沟。脯氨酸分解代谢由两种酶和一个中间的水解步骤组成。酪氨酸脱氢酶(PROH)催化L-脯氨酸氧化生成1-吡咯烷-5-羧酸(-1-pyrroline-5-cixylate，P5C)。P5C水解生成L-谷氨酸--半醛，再被依赖于NAD+的P5C脱氢酶(P5CDH)氧化成L-谷氨酸。这些酶与人类健康和疾病的许多方面有关，包括肿瘤抑制、高脯氨酸血症代谢障碍、精神分裂症易感性、延长寿命以及真菌和细菌病原体的毒力。在一些生物体中，PROSH和P5CDH被结合成一个单一的多肽链，称为Pro利用A(PUTA)。将代谢途径中的连续酶包装成单一蛋白质不仅意味着底物通道，而且还意味着单功能酶之间蛋白质-蛋白质相互作用的可能性。因此，脯氨酸分解代谢提供了一个很好的机会来比较底物在酶内和酶之间的通道。该项目的下一阶段建立在前一个资助周期中取得的三项主要成就的基础上：确定PUTA蛋白的第一个晶体结构，发现一种新的滞后底物通道动力学机制，以及发现单功能PROH和P5CDH酶之间酶间底物通道的第一个证据。其具体目的是(1)阐明在Puta家族中进化的底物通道的不同结构解决方案，(2)确定滞后通道机制的结构基础和保守性，以及(3)通过检测整个细胞中的Proh-P5CDH相互作用和确定干扰Pro代谢通道的表型后果来研究生物学背景下的底物通道。

 DESCRIPTION (provided by applicant): Metabolic enzymes in cells rarely function in isolation. Often their activities are coordinated by physical association with each other and cellular structures. A consequence of these associations is that metabolic intermediates do not equilibrate with the cellular milieu but rather are channeled between enzymes. Despite the widespread recognition that protein-protein interactions are ubiquitous, the mechanisms of substrate channeling remain relatively understudied and thus poorly understood. We help close this knowledge gap by exploring substrate channeling within and between the enzymes of proline catabolism. Proline catabolism comprises two enzymes and an intervening hydrolysis step. The flavoenzyme proline dehydrogenase (PRODH) catalyzes the oxidization of L-proline to 1-pyrroline-5-carboxylate (P5C). Hydrolysis of P5C yields L-glutamate--semialdehyde, which is oxidized to L-glutamate by the NAD+-dependent enzyme P5C dehydrogenase (P5CDH). These enzymes have been implicated in many aspects of human health and disease, including tumor suppression, hyperprolinemia metabolic disorders, schizophrenia susceptibility, life- span extension, and the virulence of fungal and bacterial pathogens. In some organisms, PRODH and P5CDH are combined into a single polypeptide chain known as proline utilization A (PutA). The packaging of sequential enzymes from a metabolic pathway into a single protein not only implies substrate channeling but also the possibility of protein-protein interactions between the monofunctional enzymes. Thus, proline catabolism affords an excellent opportunity to compare substrate channeling within and between enzymes. The next phase of this project builds upon three major accomplishments made during the previous funding cycle: determination of the first crystal structures of PutA proteins, discovery of a novel hysteretic substrate channeling kinetic mechanism, and uncovering the first evidence for inter-enzyme substrate channeling between monofunctional PRODH and P5CDH enzymes. The specific aims are to (1) elucidate the diverse structural solutions to substrate channeling that have evolved in the PutA family, (2) determine the structural basis and conservation of the hysteretic channeling mechanism, and (3) study substrate channeling in biological context by examining PRODH - P5CDH interactions in whole cells and determining the phenotypic consequences of disrupting proline metabolic channeling.