GO:0004359 glutaminase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004359 glutaminase activity is a molecular function defined as the catalysis of L-glutamine + H2O = L-glutamate + NH4+.
• Glutaminase activity is central to glutaminolysis, a pathway that supports energy production, redox balance, and biosynthesis in proliferating cells [3,6].
• The two main mammalian glutaminase enzymes, GLS (kidney-type) and GLS2 (liver-type), are regulated by oncogenic and metabolic signals, including Hedgehog-YAP and SUCLA2-mediated succinylation [2,6].
• Altered glutaminase activity is implicated in cancer, metabolic disorders, and immune cell function, making it a therapeutic target [1,5,7,8].
• Glutaminase activity can be measured by enzymatic assays, and its role in cells can be dissected using CRISPR knockout, point mutation, knock-in, and overexpression models.
• EDITGENE provides custom CRISPR cell models and screening services to study glutaminase activity and its downstream effects.
Description
Glutaminase activity (GO:0004359) is a fundamental enzymatic function that hydrolyzes L-glutamine to L-glutamate and ammonium. This reaction, often referred to as glutaminolysis, is a key entry point for glutamine carbon and nitrogen into central metabolism, supporting ATP production, redox homeostasis, and the synthesis of macromolecules such as nucleotides and lipids [3,6]. Because many cancer cells and activated immune cells depend on glutamine, glutaminase activity has emerged as a critical node in metabolic reprogramming and a potential therapeutic target [1,5,7,8]. Researchers study glutaminase activity to understand how cells adapt to metabolic stress, how oncogenic pathways rewire metabolism, and how to design inhibitors that selectively kill glutamine-addicted cells [2,4,6]. The enzyme is also relevant to non-cancer contexts, including adipocyte biology and metabolic health. This article provides a comprehensive overview of the definition, mechanism, key genes, regulation, disease links, and research methods for glutaminase activity, with a focus on CRISPR-based approaches for functional interrogation.
glutaminase activity At A Glance
| GO ID | GO:0004359 |
|---|---|
| GO term | glutaminase activity |
| Ontology | molecular_function |
| Synonym | glutaminase I; glutamine aminohydrolase activity; L-glutaminase activity; L-glutamine amidohydrolase activity |
| Major function | Catalysis of L-glutamine + H2O = L-glutamate + NH4+ |
| EC number | 3.5.1.2 |
| Reaction direction | Irreversible hydrolysis |
| Subcellular location | Mitochondrial matrix (for GLS and GLS2) |
| Pathway context | Glutaminolysis, nitrogen metabolism, glutamate biosynthesis |
What Is GO:0004359?
According to the Gene Ontology, glutaminase activity (GO:0004359) is defined as the catalysis of the reaction: L-glutamine + H2O = L-glutamate + NH4+. In other words, it is the enzymatic function that removes an amino group from glutamine, producing glutamate and ammonium. This activity is synonymous with glutaminase I, glutamine aminohydrolase activity, L-glutaminase activity, and L-glutamine amidohydrolase activity.
Why Is glutaminase activity Important in Cell Biology?
Glutaminase activity is a metabolic hub that fuels the tricarboxylic acid (TCA) cycle, supports glutathione synthesis, and maintains redox balance in rapidly dividing cells [3,6]. Its dysregulation is linked to cancer progression, immune cell activation, and metabolic disorders, making it a high-priority target for both basic research and drug development [1,5,7,8].
• Provides glutamate for glutathione synthesis and redox homeostasis [3,6].
• Supports ATP production via anaplerosis into the TCA cycle [3,6].
• Essential for activation of CD8 T cells in certain tumor contexts.
• Promotes tumor cell survival under glucose deprivation.
• Regulates hepatic stellate cell activation in liver fibrosis.
• Modulates adipocyte energy expenditure and metabolic health.
• Influences cytotoxicity of L-asparaginases in leukemia cells.
• Linked to oxidative stress response via SUCLA2-mediated succinylation.
• Potential biomarker in bladder cancer with mitochondrial reprogramming.
• Target for small-molecule inhibitors in cancer therapy [2,5,8].
Molecular Mechanism of glutaminase activity
Substrate Binding and Catalysis
In simple terms: Glutaminase grabs glutamine and splits it into glutamate and ammonia.
Glutaminase catalyzes the hydrolysis of L-glutamine to L-glutamate and ammonium. The enzyme binds glutamine in its active site, where a water molecule attacks the amide bond, releasing ammonia and forming glutamate. This reaction is irreversible and requires no cofactors.
Enzyme Isoforms and Localization
In simple terms: There are two main versions of the enzyme in humans, GLS and GLS2, which work in different tissues.
Mammalian glutaminase exists as two distinct genes: GLS (kidney-type glutaminase) and GLS2 (liver-type glutaminase). Both are mitochondrial enzymes, but they differ in tissue distribution, kinetic properties, and regulation. GLS is widely expressed and often upregulated in cancers, while GLS2 is more restricted to liver and brain [3,6].
Regulation by Succinylation and Oxidative Stress
In simple terms: A chemical tag called succinylation can turn the enzyme on or off, especially when cells are stressed.
SUCLA2, a TCA cycle enzyme, regulates GLS activity through succinylation. Under oxidative stress, changes in succinyl-CoA levels alter GLS succinylation, modulating its activity to counteract stress. This provides a direct link between mitochondrial metabolism and glutaminase function.
Signaling Pathways Controlling Glutaminase
In simple terms: Signals from genes like Hedgehog and YAP can boost glutaminase activity.
The Hedgehog-YAP signaling pathway regulates glutaminolysis by controlling glutaminase expression and activity in hepatic stellate cells. In cancer, oncogenic pathways such as STK11/LKB1 loss can alter glutaminase dependence, affecting T cell activation. These signaling inputs allow cells to adapt glutaminase activity to their metabolic needs [5,6].
Role in Glutaminolysis and Metabolic Flux
In simple terms: Glutaminase is the first step in a pathway that turns glutamine into energy and building blocks.
Glutaminase initiates glutaminolysis, converting glutamine to glutamate, which can then be further metabolized to alpha-ketoglutarate to feed the TCA cycle [3,6]. This flux supports ATP production, lipid synthesis, and nucleotide biosynthesis. In some tumors, glutaminase activity is essential for survival under glucose deprivation.
Key Genes Involved in GO:0004359 glutaminase activity
The following genes encode proteins with glutaminase activity or directly regulate it, and they are frequently studied in metabolic research.
| Gene | Major Role | Research Relevance |
|---|---|---|
| GLS | Kidney-type glutaminase; catalyzes glutamine to glutamate | Upregulated in many cancers; target for inhibitors [3,5,8] |
| GLS2 | Liver-type glutaminase; catalyzes glutamine to glutamate | Tumor suppressor in some contexts; regulates redox balance [3,6] |
| SUCLA2 | Succinyl-CoA ligase subunit; regulates GLS succinylation | Links TCA cycle to glutaminase activity under oxidative stress |
| STK11 | Serine/threonine kinase; regulates metabolic stress responses | Loss alters glutaminase dependence in lung cancer |
| YAP1 | Transcriptional co-activator; regulates glutaminolysis | Controls glutaminase expression in hepatic stellate cells |
| GLI1 | Hedgehog pathway transcription factor | Regulates glutaminolysis genes including GLS |
| MYC | Oncogenic transcription factor | Drives glutaminase expression in cancers |
| HIF1A | Hypoxia-inducible factor | Regulates glutamine metabolism under low oxygen |
| MT-CO2 | Mitochondrial cytochrome c oxidase II | Promotes glutaminolysis for survival upon glucose deprivation |
| GLS2 variant | Liver-type glutaminase isoform | Associated with metabolic health and adipocyte function |
| GOT1 | Aspartate aminotransferase | Downstream of glutaminase in glutaminolysis |
| GOT2 | Mitochondrial aspartate aminotransferase | Downstream of glutaminase in glutaminolysis |
| GLUD1 | Glutamate dehydrogenase | Converts glutamate to alpha-ketoglutarate |
| ASNS | Asparagine synthetase | Related to glutamine metabolism and L-asparaginase sensitivity |
| SLC1A5 | Glutamine transporter | Supplies glutamine for glutaminase |
| SLC7A11 | Cystine/glutamate antiporter | Linked to redox balance and glutaminase activity |
| KEAP1 | Oxidative stress regulator | Mutated in cancers with altered glutamine metabolism |
| NFE2L2 | Nrf2 transcription factor | Regulates antioxidant response and glutaminase interplay |
How Is glutaminase activity Regulated?
Glutaminase activity is regulated at multiple levels. Transcriptionally, oncogenes such as MYC and HIF1A increase GLS expression. Post-translationally, succinylation by SUCLA2 modulates GLS activity in response to oxidative stress. Signaling pathways including Hedgehog-YAP control glutaminase expression in specific cell types. Additionally, the availability of glutamine transporters and the metabolic state of the cell influence flux through glutaminase.
glutaminase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| GLS | Cancer (multiple types), glutamine addiction | CRISPR knockout in cancer cell lines; xenograft models |
| GLS2 | Metabolic disorders, redox balance | Knock-in of point mutations; overexpression in adipocytes |
| SUCLA2 | Oxidative stress response, cancer | Point mutation of succinylation sites; KO in tumor cells |
| STK11 | Lung cancer, immune evasion | KO in lung cancer cells; co-culture with T cells |
| MT-CO2 | Tumor survival under glucose deprivation | Overexpression and KO in cancer cells; metabolic assays |
Cancer Metabolism and Glutamine Addiction
Many cancer cells exhibit increased glutaminase activity to support rapid proliferation and survival [3,6]. GLS is upregulated in various tumors, and its inhibition impairs tumor growth in preclinical models [2,5,8]. In bladder cancer, mitochondrial reprogramming via glutamine metabolism is associated with patient outcomes. Glutaminase activity also determines sensitivity to L-asparaginase in leukemia cell lines.
Metabolic Disorders and Adipocyte Biology
Reduced adipocyte glutaminase activity promotes energy expenditure and improves metabolic health, suggesting a role in obesity and insulin resistance. This highlights glutaminase as a potential target for metabolic diseases beyond cancer.
Immune Cell Function and Tumor Microenvironment
Glutaminase inhibition impairs CD8 T cell activation in STK11/LKB1-deficient lung cancer, indicating that glutaminase activity is critical for anti-tumor immunity. This creates a therapeutic challenge: targeting glutaminase may also affect immune responses.
Liver Fibrosis and Hepatic Stellate Cells
Hedgehog-YAP signaling regulates glutaminolysis to control hepatic stellate cell activation, a key step in liver fibrosis. Modulating glutaminase activity may therefore influence fibrotic disease progression.
From glutaminase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of GLS affect tumor growth? | GLS knockout cell lines and mouse xenografts |
| How does succinylation regulate GLS activity? | Point mutation of lysine residues in GLS (succinylation sites) |
| What is the effect of a disease-associated GLS variant? | Knock-in of the variant using CRISPR |
| Where is glutaminase localized in live cells? | Tagged knock-in of GLS with fluorescent protein |
| Does overexpression of GLS2 alter metabolism? | Overexpression of GLS2 in cell lines |
| Can glutaminase activity be rewired by oncogenes? | CRISPR activation or knockout of signaling genes (e.g., STK11) |
How to Study the glutaminase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Glutaminase activity assay | Enzyme activity via glutamate/ammonia production | Validation of KO or inhibitor efficacy |
| 13C-glutamine tracing | Metabolic flux through glutaminolysis | Quantifying pathway activity in cells |
| CRISPR knockout screening | Gene essentiality and synthetic lethality | Identifying modifiers of glutaminase dependence |
| Western blot | Protein expression levels | Confirming KO or overexpression |
| Immunofluorescence | Subcellular localization | Visualizing mitochondrial glutaminase |
| RNA-seq | Transcriptional changes | Assessing pathway rewiring after perturbation |
| Seahorse assay | Oxygen consumption and glycolysis | Measuring metabolic phenotype |
Enzymatic Activity Assays
Glutaminase activity can be measured directly using colorimetric or fluorometric assays that detect glutamate or ammonia production. These assays are used to validate CRISPR knockouts or inhibitor effects.
Metabolic Flux Analysis
Isotope tracing with 13C-glutamine followed by mass spectrometry quantifies flux through glutaminase and downstream pathways [3,6]. This method reveals how genetic perturbations alter glutaminolysis.
CRISPR Screening and Functional Genomics
Genome-wide CRISPR knockout screens can identify genes that modulate sensitivity to glutaminase inhibitors or glutamine deprivation [5,8]. Such screens uncover synthetic lethal interactions and resistance mechanisms.
Proteomics and Post-Translational Modification Analysis
Mass spectrometry-based proteomics can detect succinylation and other modifications on GLS, as shown for SUCLA2-mediated regulation. This helps map regulatory sites and understand signaling.
How CRISPR Can Be Used to Study GO:0004359 glutaminase activity
Knockout
CRISPR knockout of GLS or GLS2 eliminates glutaminase activity, allowing researchers to test its requirement for cell proliferation, survival, and metabolic flux [5,8]. Knockout cell lines are also used to validate inhibitor specificity.
Point Mutation
Introducing point mutations in GLS at succinylation sites or catalytic residues can dissect the contribution of specific modifications to enzyme activity. Such models help distinguish catalytic function from regulatory interactions.
Knock-in
Knock-in of disease-associated variants or tagged versions of GLS enables real-time tracking of enzyme localization and dynamics. This approach can also create isogenic models to study variant effects on glutaminase activity.
Overexpression
Overexpression of GLS or GLS2 via CRISPR activation or lentiviral delivery increases glutaminase activity, mimicking the upregulated state seen in cancers [3,6]. These models are useful for testing metabolic dependencies and drug responses.
How EDITGENE Supports glutaminase activity Research
Researchers studying glutaminase activity-related genes often need to determine whether a candidate gene is causally involved in metabolic reprogramming, disease progression, or therapeutic response. EDITGENE provides a comprehensive suite of CRISPR services to generate precisely engineered cell models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for glutaminase activity research.
Frequently Asked Questions About glutaminase activity
What is glutaminase activity?
Glutaminase activity (GO:0004359) is the enzymatic catalysis of L-glutamine to L-glutamate and ammonium, a key step in glutaminolysis.
What genes are involved in glutaminase activity?
The main genes are GLS and GLS2, which encode kidney-type and liver-type glutaminase, respectively. Regulators include SUCLA2, STK11, and YAP1 [2,3,5,6].
What is the GO ID for glutaminase activity?
The Gene Ontology ID for glutaminase activity is GO:0004359.
How is glutaminase activity regulated?
It is regulated transcriptionally by MYC and HIF1A, post-translationally by succinylation via SUCLA2, and through signaling pathways like Hedgehog-YAP [2,3,6].
What diseases are associated with glutaminase activity?
Altered glutaminase activity is linked to cancer, metabolic disorders, liver fibrosis, and immune dysfunction [1,4,5,6,7,8].
How can I measure glutaminase activity in the lab?
Common methods include enzymatic assays detecting glutamate or ammonia, and isotope tracing with 13C-glutamine followed by mass spectrometry [3,6].
What are the synonyms for glutaminase activity?
Synonyms include glutaminase I, glutamine aminohydrolase activity, L-glutaminase activity, and L-glutamine amidohydrolase activity.
Which cell models are best for studying glutaminase activity?
CRISPR knockout, point mutation, knock-in, and overexpression models in relevant cell lines (e.g., cancer, adipocytes) are widely used [1,2,5,8].
Is glutaminase a good drug target?
Yes, glutaminase inhibitors are being explored for cancer therapy, though effects on immune cells must be considered [2,5,8].
What is the difference between GLS and GLS2?
GLS is kidney-type and often upregulated in cancers, while GLS2 is liver-type and has context-dependent roles, sometimes tumor suppressive [3,6].
Conclusion
Glutaminase activity (GO:0004359) is a central metabolic function that supports cell growth, redox balance, and survival. Its dysregulation contributes to cancer, metabolic diseases, and immune responses, making it a compelling target for therapeutic intervention. Understanding its regulation and downstream effects requires robust experimental models, and CRISPR-based approaches offer precise tools to dissect gene function. EDITGENE provides end-to-end services to generate and analyze such models, empowering researchers to advance glutaminase biology.
References
- 1. Lecoutre S et al.. 2024. Reduced adipocyte glutaminase activity promotes energy expenditure and metabolic health.. Nat Metab 6(7):1329-1346 PMID: 39009762
- 2. Tong Y et al.. 2021. SUCLA2-coupled regulation of GLS succinylation and activity counteracts oxidative stress in tumor cells.. Mol Cell 81(11):2303-2316.e8 PMID: 33991485
- 3. Curthoys NP et al.. 1995. Regulation of glutaminase activity and glutamine metabolism.. Annu Rev Nutr 15:133-59 PMID: 8527215
- 4. Parmentier JH et al.. 2015. Glutaminase activity determines cytotoxicity of L-asparaginases on most leukemia cell lines.. Leuk Res 39(7):757-62 PMID: 25941002
- 5. Best SA et al.. 2022. Glutaminase inhibition impairs CD8 T cell activation in STK11-/Lkb1-deficient lung cancer.. Cell Metab 34(6):874-887.e6 PMID: 35504291
- 6. Du K et al.. 2018. Hedgehog-YAP Signaling Pathway Regulates Glutaminolysis to Control Activation of Hepatic Stellate Cells.. Gastroenterology 154(5):1465-1479.e13 PMID: 29305935
- 7. Kami Reddy KR et al.. 2024. Mitochondrial reprogramming by activating OXPHOS via glutamine metabolism in African American patients with bladder cancer.. JCI Insight 9(17) PMID: 39253977
- 8. Yi Y et al.. 2025. Mitochondrial-cytochrome c oxidase II promotes glutaminolysis to sustain tumor cell survival upon glucose deprivation.. Nat Commun 16(1):212 PMID: 39747079
