Mechanisms of glutamate dehydrogenase allostery

描述（由申请人提供）：谷氨酸脱氢酶（GDH）是一种在所有生物体中发现的古老酶。由这种酶催化的谷氨酸可逆氧化脱氨为2-酮戊二酸的基本化学在各个时期保持不变。然而，来自动物界的GDH由大量化合物变构调节，而来自其他界的酶完全不受调节。通过我们的个人和合作的努力，我们已经确定了动物GDH与这些变构调节剂中的几种复合的结构，并具有如何进行变构的模型。我们还发现影响GTP抑制的突变是儿童高胰岛素血症/高血氨症（HI/HA）综合征的根本原因，这表明了GDH调节的重要性。此外，我们已经表明GDH参与葡萄糖和亮氨酸刺激的正常胰腺组织胰岛素分泌。我们认为，这种胰岛素稳态的调节是由于GDH介导的细胞内信号分子谷氨酰胺的控制。 因此，很明显，了解GDH的变构调节对了解胰岛素稳态至关重要。为此，该提案旨在使用技术组合来了解这些各种调节剂的结合位置以及它们如何调节酶活性。我们还将进一步分析一些HI/HA突变体，以更好地了解这种危及生命的疾病的病理学。也许最令人兴奋的是，我们已经利用我们对GDH的理解发现了一个新的无毒化合物家族，这些化合物通过GDH起作用，并可能治疗HI/HA和II型糖尿病。最后，我们已经表明，来自纤毛虫的GDH是动物和其他王国之间进化上的“缺失环节”。通过进一步研究这种形式的GDH，我们将通过更好地理解它是如何进化的以及为什么进化来阐明动物变构。总之，这些研究不仅将回答蛋白质亚基在变构过程中如何相互交流的基本问题，而且将巩固我们的假设，即GDH的变构调节在胰岛素稳态中起着关键作用。

DESCRIPTION (provided by applicant): Glutamate dehydrogenase (GDH) is an ancient enzyme found in all living organisms. The underlying chemistry of the reversible oxidative deamination of glutamate to 2-oxoglutarate catalyzed by this enzyme has remained unchanged through the epochs. However, GDH from the animal kingdom is allosterically regulated by a large number compounds while the enzyme from other kingdoms is completely unregulated. Through our individual and collaborative efforts, we have determined the structures of animal GDH complexed with several of these allosteric regulators and have models for how allostery is exacted. We have also shown the importance of GDH regulation by our finding that mutations that affect GTP inhibition are the root cause of the hyperinsulinism/hyperammonemia (HI/HA) syndrome in children. Furthermore, we have shown that GDH is involved in both glucose and leucine stimulated secretion of insulin from normal pancreatic tissue. We propose that this regulation of insulin homeostasis is due to GDH-mediated control of the intracellular signaling molecule, glutamine. It is therefore apparent that understanding the allosteric regulation of GDH is crucial to understanding insulin homeostasis. To that end, this proposal aims to use a combination of techniques to understand where these various regulators bind and how they modulate enzymatic activity. We will also further analyze some HI/HA mutants to better understand the pathology of this life-threatening disorder. Perhaps most exciting is that we have leveraged our understanding of GDH to discover a family of new, non-toxic compounds that act via GDH and can potentially treat both HI/HA and type II diabetes. Finally, we have shown that the GDH from Ciliates is an evolutionary 'missing link' between animals and the other kingdoms. By further studying this form of GDH we will elucidate animal allostery by better understanding how and why it evolved. Together, these studies will not only answer fundamental questions as to how protein subunits communicate with each other during allostery but will solidify our hypothesis that allosteric regulation of GDH plays a critical role in insulin homeostasis.