Coordination of functions by proline metabolic proteins.

 描述（由申请人提供）：细胞中的代谢酶很少单独发挥作用。通常，它们的活动通过彼此和细胞结构的物理关联来协调。这些关联的结果是代谢中间产物不与细胞环境平衡，而是在酶之间形成通道。尽管普遍认为蛋白质-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研究不足，因此知之甚少。我们通过探索脯氨酸催化剂酶内和酶间的底物通道来帮助缩小这一知识差距。脯氨酸催化剂包括两种酶和一个插入的水解步骤。黄素酶脯氨酸脱氢酶（PRODH）催化L-脯氨酸氧化为β 1-吡咯啉-5-羧酸（P5 C）。P5 C的水解产生L-谷氨酸-β-半醛，其被NAD+依赖性酶P5 C脱氢酶（P5 CDH）氧化为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、高脯氨酸血症代谢紊乱、精神分裂症易感性、寿命延长以及真菌和细菌病原体的毒力。在某些生物体中，PRODH和P5 CDH结合成一条多肽链，称为脯氨酸利用A（PutA）。将来自代谢途径的连续酶包装成单个蛋白质不仅意味着底物通道，而且还意味着单功能酶之间的蛋白质-蛋白质相互作用的可能性。因此，脯氨酸催化剂提供了一个很好的机会，比较内和酶之间的底物通道。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，并发现单功能PRODH和P5 CDH酶之间酶间底物通道的第一个证据。具体目的是（1）阐明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）通过检查全细胞中的PRODH-P5 CDH相互作用并确定破坏脯氨酸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.