Glutamate dehydrogenase (GDH) is an enzyme that catalyzes the oxidative deamination of glutamate, releasing 2-oxoglutarate, NH3, and NAD(P)H in a bisubstrate NAD(P)+-dependent reaction. It also facilitates the backward reaction of 2-oxoglutarate reductive amination to glutamate. In animals, both NAD+ and NADP+ serve as electron acceptors. The direction of the GDH reaction varies in different cell and tissue types. In the rat brain, oxidative deamination of glutamate predominates, although neuronal GDH activity is lower than in astrocytes. In the liver, the reaction is in equilibrium, while in the pancreas and kidney, it catalyzes oxidative deamination.
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Intracellular Localization of GDH in Animals
In animals, GDH was traditionally thought to be exclusively localized in mitochondria, serving as a marker for these organelles. Its activity in the mitochondrial matrix can be influenced by interactions with anionic phospholipids like cardiolipin and phosphatidylserine on the inner mitochondrial membrane, potentially affecting its binding to small molecules such as estrogens. However, recent studies have identified a distinct pool of GDH associated with the nuclear fraction, suggesting its presence in both mitochondria and the nucleus. These nuclear and mitochondrial forms exhibit different solubilization properties and kinetic characteristics. Moreover, GDH has been found in the granular endoplasmic reticulum of rat liver and in lysosomes and endosomes, where it displays tubulin-binding activity. Additionally, GDH has been observed in association with cytoskeletal elements like GFAP protein in human astrocytes.
Researchers have also discovered membrane-bound forms of GDH, resistant to extraction by certain detergents but solubilized by others, indicating their presence in various cellular membranes. Soluble forms of GDH with differing thermostability and allosteric regulation have been identified in rat brain, suggesting functional diversity. Furthermore, GDH has been detected in the cytosol of bovine thyroid glands, where it likely participates in thyroid hormone synthesis. These findings highlight the diverse intracellular localization and functional roles of GDH across different animal tissues and organelles.
Structural Insights into GDH
Mitochondrial GDH Structure
In mammals, mitochondrial GDH forms a homohexamer, with each subunit weighing approximately 56 kDa. It comprises catalytic, NAD(P)+-binding, and regulatory domains. The core structure includes two trimeric N-terminal glutamate-binding domains, predominantly arranged as β-strands. Adjacent to these are NAD+-binding domains, with rotating pivot helix regions implicated in catalysis. Notably, trimeric "antennae" extend from the NAD+-binding domains, facilitating conformational changes and intersubunit communication, influencing negative cooperativity and allosteric regulation. Although absent in GDH from other species like Neurospora sp. or Clostridia sp., these GDHs exhibit allosteric regulation through alternative structural elements.
Fig. 1 Structure of glutamate dehydrogenase (Smith H.Q., et al. 2019).
Conformational Dynamics
Recent cryoelectron microscopy studies revealed the open and closed conformations of bovine GDH, shedding light on the mobility of catalytic NAD+-binding domains and their impact on ADP-dependent activation. The structural flexibility of GDH catalytic and allosteric sites suggests a mechanism for ADP-dependent catalysis. Moreover, emerging research identifies novel binding sites for green tea polyphenols and analogs, adding to the complexity of GDH regulation.
Sequence Variations
Sequence alignment of human GDH isoenzymes reveals significant differences in leader peptides despite a high sequence identity. These variations influence mitochondrial localization, with substitutions like Glu7Lys, Asp25His, and Trp32Arg affecting charge and delivery to mitochondria. Additionally, post-translational modifications such as phosphorylation of Ser227 and Ser384 differ between GDH1 and GDH2, suggesting distinct regulatory mechanisms. Evolutionary trends, illustrated by Ser384/Thr substitution, highlight species-specific adaptations within the hominoid taxonomic clade. Other modifications like N6-malonylation and Tyr512 phosphorylation further contribute to the diversity of GDH regulation.
Post-Translational Modifications of GDH
NAD+-Dependent ADP-Ribosylation
GDH undergoes NAD+-dependent ADP-ribosylation, catalyzed by sirtuin 4 (SIRT4), resulting in enzyme inactivation. This modification involves cleavage of NAD+ with ADP-ribose transferred to a conserved cysteine residue in GDH, leading to loss of catalytic activity. Reactivation of ADP-ribosylated GDH occurs via Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. This modification potentially plays a role in cellular nitrogen metabolism regulation.
Phosphorylation
GDH phosphorylation has been observed in various animal species, impacting enzyme activity and metabolic pathways. For instance, phosphorylation in snails during hibernation increases glutamate deamination activity, altering nitrogen metabolism. Crayfish GDH phosphorylation under hypoxia stimulates reductive amination of 2-oxoglutarate, affecting ATP production and tricarboxylic acid cycle activity. However, phosphorylation in gophers during hibernation shifts metabolism towards gluconeogenesis. Although multiple phosphorylation sites have been identified in mammalian GDHs, their specific functional significance remains unclear.
Lysine and Cysteine Acylation
GDH undergoes acetylation, succinylation, malonylation, and fatty acid acylation at lysine and cysteine residues. Acetylation affects subunit interactions, altering allosteric regulation. Regulation of GDH acetylation is mediated by mitochondrial acetyltransferases and deacetylases like SIRT3. Succinylation and malonylation, catalyzed by yet unidentified enzymes, also modulate GDH function. These acylations may influence GDH reactivity to allosteric regulators, potentially affecting metabolic regulation.
Oxidation and Nitration
Oxidation and nitration of GDH significantly impact enzyme activity and are implicated in various diseases. Nitrating agents can inactivate GDH, with specific tyrosine residues targeted depending on the agent. Tyrosine nitration can either restore or further decrease GDH activity, depending on the context. These modifications may alter GDH's response to allosteric regulators, potentially influencing metabolic pathways and signaling processes.
Medical Implications of GDH
Peripheral Tissues
GDH plays vital roles in various organs. In kidneys, it helps regulate NH4+ ion concentration, while in pancreatic β-cells, it influences insulin secretion. Mutations in GLUD1 affecting GDH sensitivity to GTP can lead to hyperinsulinism/hyperammonemia (HI/HA) syndrome, impacting glucose and NH4+ levels. Similarly, mutations in short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), inhibiting GDH binding, contribute to inappropriate insulin release. ADP-ribosylation of GDH by SIRT4 modulates insulin production, highlighting its regulatory significance under different metabolic conditions.
Brain GDH
Dysregulation of GDH activity in the brain is implicated in various neurodegenerative diseases. Both decreased and up-regulated GDH activity can be damaging. GDH deficiency is observed in conditions like multisystemic atrophy, leading to increased glutamate levels and excitotoxicity. Conversely, GDH up-regulation, as seen in diseases like HI/HA syndrome, results in neurotransmitter depletion and neurological symptoms. Gain-of-function mutations in the GLUD2 gene may contribute to early onset Parkinson's disease. Furthermore, GDH overexpression is associated with neuronal loss and the development of conditions like Alzheimer's disease and schizophrenia, suggesting its involvement in multiple neurodegenerative processes.
Malignant Transformation
Altered glutamine metabolism, including up-regulation of GDH activity, is associated with tumor growth and poor prognosis in various cancers. Inhibition of SIRT4 can increase GDH activity, promoting tumor growth. In glioma cells, GDH expression enables survival under glucose deprivation, highlighting its role as an alternative carbon source. Targeting GDH activity, either through siRNA or novel inhibitors, shows promise in inhibiting cancer cell proliferation. Additionally, GDH is involved in cancer redox homeostasis and may contribute to glutamine dependence in tumor cells. Understanding the interplay between glucose and glutamate metabolism in cancer cells is crucial for developing effective cancer therapies.
Conclusion
Glutamate dehydrogenase is a multifaceted enzyme at the intersection of cellular metabolism and signaling pathways. Its activity is finely tuned by nucleotide-dependent regulation and post-translational modifications, which govern metabolic flux and cellular responses to environmental cues. Further elucidation of GDH regulation and function promises to uncover novel therapeutic strategies for metabolic disorders and diseases associated with dysregulated metabolism.
References
- Smith H.Q., et al. Glutamate dehydrogenase, a complex enzyme at a crucial metabolic branch point. Neurochemical Research. 2019, 44: 117-32.
- Bunik V., et al. Multiple forms of glutamate dehydrogenase in animals: structural determinants and physiological implications. Biology. 2016, 5(4): 53.
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