GO:0004774 succinate-CoA ligase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004774 succinate-CoA ligase activity catalyzes the reversible conversion of succinate, CoA, and a nucleotide triphosphate into succinyl-CoA, a nucleotide diphosphate, and phosphate.
• This activity is essential for the mitochondrial tricarboxylic acid (TCA) cycle, where it links succinyl-CoA production to substrate-level phosphorylation via either ADP- or GDP-forming isoforms.
• Mutations in SUCLA2 and SUCLG1, encoding the β- and α-subunits of succinate-CoA ligase, cause mitochondrial DNA depletion syndromes and Leigh-like encephalomyopathies [1,7].
• SUCLA2 and SUCLG1 are also implicated in cancer progression, where they regulate succinylation of glutaminase (GLS) and stress granule assembly to influence redox balance and metastasis [3,4].
• Recent studies show that succinate-CoA ligase activity is modulated by metabolic signals such as β-hydroxybutyrylation and glutaminolysis, affecting obesity and Alzheimer's disease pathologies [2,6].
• CRISPR-based knockout, point mutation, and knock-in models are powerful tools to dissect the tissue-specific roles of succinate-CoA ligase subunits in health and disease.
Description
Succinate-CoA ligase activity (GO:0004774) is a fundamental enzymatic function in mitochondrial energy metabolism, catalyzing the reversible formation of succinyl-CoA from succinate and coenzyme A coupled to the hydrolysis of a nucleotide triphosphate. This reaction occupies a central position in the tricarboxylic acid (TCA) cycle, where it represents the only step that generates high-energy phosphate bonds through substrate-level phosphorylation, thereby directly contributing to cellular ATP or GTP pools. In humans, succinate-CoA ligase exists as two heterodimeric isoforms: one using ADP (SUCLA2-SUCLG1) and one using GDP (SUCLG2-SUCLG1), each composed of a shared α-subunit and a distinct β-subunit [1,7]. The activity is critical for maintaining mitochondrial nucleotide homeostasis and for providing succinyl-CoA for heme synthesis and ketone body metabolism. Beyond its housekeeping role, succinate-CoA ligase activity has emerged as a regulatory node in cancer, metabolic disorders, and neurodegeneration. For example, SUCLA2 loss alters glutamine metabolism and redox balance in tumor cells, while SUCLG1 mutations lead to severe mitochondrial DNA depletion syndromes [3,4,7]. Recent work has also linked the enzyme to β-hydroxybutyrylation and obesity-associated macrophage polarization, underscoring its broad physiological relevance [2,6]. Understanding the molecular mechanisms, regulation, and disease connections of succinate-CoA ligase activity is therefore essential for researchers in metabolism, oncology, and rare mitochondrial diseases.
succinate-CoA ligase activity At A Glance
| GO ID | GO:0004774 |
|---|---|
| GO term | succinate-CoA ligase activity |
| Ontology | molecular_function |
| Synonym | none |
| Major function | Catalyzes the reversible conversion of succinate, CoA, and a nucleotide triphosphate to succinyl-CoA, a nucleotide diphosphate, and phosphate |
| Reaction direction | Reversible; can synthesize succinyl-CoA or generate succinate and nucleotide triphosphate |
| Nucleotide specificity | ATP- or GTP-dependent isoforms exist (ADP-forming SUCLA2 and GDP-forming SUCLG2) |
| Subcellular location | Mitochondrial matrix |
| Pathway context | Tricarboxylic acid (TCA) cycle; substrate-level phosphorylation |
What Is GO:0004774?
Succinate-CoA ligase activity (GO:0004774) is defined as the catalysis of the reaction: succinate + CoA + nucleotide triphosphate = nucleotide diphosphate + phosphate + succinyl-CoA. This reversible reaction couples the cleavage of a nucleotide triphosphate (ATP or GTP) to the formation of the thioester bond in succinyl-CoA, simultaneously generating a nucleotide diphosphate and inorganic phosphate. The enzyme operates in the mitochondrial matrix and is essential for the TCA cycle, where it facilitates the interconversion of succinate and succinyl-CoA while supporting substrate-level phosphorylation.
Why Is succinate-CoA ligase activity Important in Cell Biology?
Succinate-CoA ligase activity is indispensable for mitochondrial energy transduction and biosynthetic processes. By catalyzing the only substrate-level phosphorylation step in the TCA cycle, it directly contributes to cellular nucleotide pools and maintains flux through the cycle. Its products, succinyl-CoA and succinate, serve as precursors for heme biosynthesis, ketone body utilization, and signaling molecules that influence epigenetic states and redox homeostasis [1,3]. Dysregulation of this activity is linked to severe mitochondrial diseases, cancer progression, and metabolic disorders, making it a compelling target for both basic research and therapeutic development [1,2,4,7].
• Provides a key substrate-level phosphorylation step in the TCA cycle, generating ATP or GTP.
• Supplies succinyl-CoA for heme synthesis and ketone body metabolism.
• Mutations in SUCLA2 and SUCLG1 cause mitochondrial DNA depletion syndromes and Leigh-like encephalopathies [1,7].
• SUCLA2-mediated regulation of GLS succinylation affects redox balance and tumor growth.
• SUCLG2 promotes stress granule assembly and cancer metastasis.
• Macrophage SUCLA2 couples glutaminolysis to AMPK signaling and obesity.
• β-hydroxybutyrylation of TCA cycle enzymes, including succinate-CoA ligase, modulates Alzheimer's disease pathology.
• Tissue-specific knockout of Sucla2 in skeletal muscle recapitulates mitochondrial myopathy phenotypes.
• Valproyl-CoA inhibits succinate-CoA ligase, linking drug metabolism to mitochondrial toxicity.
• The enzyme is a potential biomarker and therapeutic target in metabolic and neoplastic diseases [2,3,4].
What Happens During succinate-CoA ligase activity?
Substrate binding and nucleotide triphosphate hydrolysis
In simple terms: The enzyme grabs succinate, CoA, and an energy molecule (ATP or GTP) and prepares to join them.
The catalytic cycle begins with the binding of succinate, coenzyme A, and a nucleotide triphosphate (ATP for SUCLA2-containing isoforms or GTP for SUCLG2-containing isoforms) to the enzyme's active site. The α-subunit (SUCLG1) provides the nucleotide-binding pocket, while the β-subunit (SUCLA2 or SUCLG2) confers specificity for the nucleotide and succinate. Upon binding, the nucleotide triphosphate is hydrolyzed, releasing energy that drives the subsequent chemical steps.
Formation of succinyl-CoA and release of products
In simple terms: The energy from the nucleotide is used to attach CoA to succinate, forming succinyl-CoA, and the leftover pieces are released.
The hydrolysis of the nucleotide triphosphate is coupled to the formation of a thioester bond between succinate and CoA, yielding succinyl-CoA, a nucleotide diphosphate, and inorganic phosphate. This reaction is reversible; under conditions of high succinyl-CoA, the enzyme can catalyze the reverse reaction to generate succinate and a nucleotide triphosphate, thereby buffering mitochondrial succinyl-CoA levels. The reaction is essential for maintaining TCA cycle flux and for providing succinyl-CoA for anabolic pathways.
Role in substrate-level phosphorylation and energy balance
In simple terms: This step directly makes a high-energy phosphate molecule, helping the cell store energy.
The succinate-CoA ligase reaction is the only step in the TCA cycle that generates a high-energy phosphate bond through substrate-level phosphorylation. In the forward direction (succinyl-CoA to succinate), the energy from succinyl-CoA thioester hydrolysis is used to phosphorylate a nucleotide diphosphate to a triphosphate (e.g., ADP to ATP or GDP to GTP). This directly contributes to mitochondrial and cellular energy pools, linking TCA cycle activity to nucleotide homeostasis.
Integration with glutamine metabolism and redox regulation
In simple terms: The enzyme also helps control how cells use glutamine and manage oxidative stress.
Recent studies have revealed that SUCLA2, the ADP-forming β-subunit, interacts with and regulates glutaminase (GLS) through succinylation, thereby influencing glutamine metabolism and redox balance in tumor cells. Loss of SUCLA2 leads to increased GLS activity and altered reactive oxygen species (ROS) levels, which can promote cancer cell survival or death depending on context. This crosstalk highlights that succinate-CoA ligase activity extends beyond classical TCA cycle functions to modulate cellular stress responses [3,4].
Key Genes Involved in GO:0004774 succinate-CoA ligase activity
The following genes encode subunits and regulators of succinate-CoA ligase activity, with diverse roles in mitochondrial metabolism and disease.
| Gene | Major Role | Research Relevance |
|---|---|---|
| SUCLG1 | Encodes the α-subunit shared by both ADP- and GDP-forming succinate-CoA ligase isoforms | Mutations cause mitochondrial DNA depletion syndrome and Leigh-like encephalopathy [1,7] |
| SUCLA2 | Encodes the ADP-forming β-subunit | Mutations cause mitochondrial DNA depletion syndrome; regulates GLS succinylation and cancer redox [1,3] |
| SUCLG2 | Encodes the GDP-forming β-subunit | Promotes stress granule assembly and cancer metastasis |
| GLS | Glutaminase; regulated by SUCLA2-mediated succinylation | Influences redox balance and tumor growth |
| AMPK | Energy sensor kinase; modulated by macrophage SUCLA2 | Links succinate-CoA ligase activity to obesity and metabolic signaling |
| LDHA | Lactate dehydrogenase A; may be affected by TCA cycle flux | Potential downstream target in metabolic reprogramming |
| SDHA | Succinate dehydrogenase complex flavoprotein subunit A | TCA cycle enzyme adjacent to succinate-CoA ligase; used as reference in metabolic studies |
| SDHB | Succinate dehydrogenase complex iron sulfur subunit B | TCA cycle enzyme; context for succinate metabolism |
| IDH3A | Isocitrate dehydrogenase 3 subunit alpha | TCA cycle enzyme; provides isocitrate for succinyl-CoA production |
| OGDH | Oxoglutarate dehydrogenase | Produces succinyl-CoA, the substrate for succinate-CoA ligase |
| DLST | Dihydrolipoamide S-succinyltransferase | Component of OGHD complex; upstream of succinate-CoA ligase |
| ACAT1 | Acetyl-CoA acetyltransferase 1 | Ketone body metabolism; intersects with succinyl-CoA utilization |
| OXCT1 | 3-oxoacid CoA transferase 1 | Ketone body utilization; uses succinyl-CoA as CoA donor |
| HMGCS2 | 3-hydroxy-3-methylglutaryl-CoA synthase 2 | Ketogenesis; competes for acetyl-CoA and succinyl-CoA pools |
| SIRT3 | NAD-dependent deacetylase sirtuin-3 | Regulates mitochondrial enzyme acetylation, including TCA cycle enzymes |
| SIRT5 | NAD-dependent desuccinylase sirtuin-5 | Removes succinyl groups from lysine residues, counteracting succinylation |
| GLS2 | Glutaminase 2 | Liver-specific glutaminase; may be regulated by succinylation |
| NLRP3 | NLR family pyrin domain containing 3 | Inflammasome; linked to macrophage metabolic state |
How Is succinate-CoA ligase activity Regulated?
Succinate-CoA ligase activity is regulated at multiple levels. Transcriptionally, the expression of SUCLA2 and SUCLG1 is controlled by mitochondrial biogenesis regulators such as PGC-1α and TFAM, although direct evidence in the context of this GO term is limited. Post-translationally, the enzyme is subject to lysine succinylation, which can affect its activity and interactions; SIRT5 acts as a desuccinylase that removes these marks. Additionally, β-hydroxybutyrylation of TCA cycle enzymes, including succinate-CoA ligase subunits, has been observed in Alzheimer's disease models, suggesting that ketone body metabolism can modulate enzyme function. Metabolically, the enzyme's activity is influenced by substrate availability (succinate, CoA, nucleotides) and by feedback from downstream pathways such as glutaminolysis and AMPK signaling [2,3].
succinate-CoA ligase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| SUCLA2 | Mitochondrial DNA depletion syndrome; Leigh-like encephalomyopathy | Sucla2 knockout mouse (skeletal muscle-specific) |
| SUCLG1 | Mitochondrial DNA depletion syndrome; fatal infantile lactic acidosis | Patient-derived fibroblasts; CRISPR knock-in of patient mutations |
| SUCLG2 | Cancer metastasis; stress granule assembly | SUCLG2 knockout cancer cell lines; xenograft models |
| SUCLA2 | Obesity; macrophage polarization | Macrophage-specific Sucla2 knockout mice |
| SUCLA2 | Alzheimer's disease; β-hydroxybutyrylation | APP/PS1 mice treated with β-hydroxybutyrate |
Mitochondrial DNA depletion syndromes and encephalomyopathies
Biallelic mutations in SUCLA2 or SUCLG1 cause severe mitochondrial DNA depletion syndromes, often presenting as Leigh-like encephalomyopathy with hypotonia, developmental delay, and lactic acidosis [1,7]. These mutations impair succinate-CoA ligase activity, leading to reduced mitochondrial nucleotide pools and subsequent mtDNA depletion. Phenotypic variability is observed, with some patients surviving into childhood while others die in infancy.
Cancer metabolism and metastasis
SUCLA2 and SUCLG2 play context-dependent roles in cancer. SUCLA2 loss alters glutamine metabolism by increasing GLS activity through reduced succinylation, which can promote tumor cell survival under oxidative stress. Conversely, SUCLG2 (the GDP-forming β-subunit) promotes stress granule assembly and drives metastasis in breast and other cancers, suggesting that specific isoforms have distinct oncogenic functions. Targeting succinate-CoA ligase activity or its downstream effectors may offer therapeutic opportunities [3,4].
Metabolic disorders and neurodegeneration
Macrophage SUCLA2 couples glutaminolysis to AMPK activation, influencing obesity-associated inflammation and systemic metabolism. In Alzheimer's disease models, β-hydroxybutyrate-induced β-hydroxybutyrylation of TCA cycle enzymes, including succinate-CoA ligase, attenuates disease-associated pathologies, suggesting a protective role for ketone body metabolism. These findings link succinate-CoA ligase activity to broader metabolic and neurodegenerative conditions [2,6].
From succinate-CoA ligase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the effect of complete loss of succinate-CoA ligase activity on mitochondrial function? | CRISPR knockout of SUCLA2 or SUCLG1 in HEK293T or HeLa cells |
| How do patient-specific point mutations affect enzyme stability and activity? | CRISPR point mutation knock-in of SUCLA2 or SUCLG1 mutations in patient fibroblasts or iPSCs |
| Can wild-type SUCLA2 rescue the phenotype of SUCLA2-deficient cells? | CRISPR knock-in of tagged SUCLA2 (e.g., FLAG) for rescue and localization studies |
| What are the tissue-specific consequences of SUCLA2 loss in muscle? | Muscle-specific Sucla2 knockout mouse |
| Does overexpression of SUCLG2 promote metastasis? | CRISPR overexpression (e.g., CRISPRa) of SUCLG2 in cancer cell lines followed by xenograft |
| How does SUCLA2 regulate GLS succinylation? | CRISPR knockout of SUCLA2 combined with GLS succinylation assays and proteomics |
How to Study the succinate-CoA ligase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzymatic activity assay | Succinate-CoA ligase catalytic rate | Validation of mutations or inhibitors |
| CRISPR knockout screen | Genes affecting cell fitness under metabolic stress | Identification of synthetic lethal partners |
| Succinylation proteomics | Lysine succinylation on target proteins | Regulation of GLS and TCA enzymes |
| 13C metabolic flux analysis | Flux through TCA cycle and substrate utilization | Metabolic reprogramming in cancer or obesity [2,6] |
| Immunoprecipitation-Western | Protein interactions and post-translational modifications | SUCLA2-GLS interaction and succinylation |
| Mitochondrial respiration assay | Oxygen consumption rate (OCR) | Functional impact of SUCLA2 loss |
| mtDNA copy number qPCR | Mitochondrial DNA depletion | Diagnosis of SUCLA2/SUCLG1-related syndromes [1,7] |
| Stress granule imaging | Assembly of stress granules | SUCLG2-mediated metastasis mechanisms |
Enzymatic activity assays
Succinate-CoA ligase activity can be measured spectrophotometrically by coupling the formation of succinyl-CoA to a downstream reaction, such as the disappearance of NADH in the presence of pyruvate kinase and lactate dehydrogenase. Alternatively, radioactive or HPLC-based methods can directly quantify succinyl-CoA or nucleotide triphosphate production. These assays are essential for validating the impact of mutations or inhibitors.
CRISPR-based genetic screens
Genome-wide CRISPR knockout screens can identify genes that modulate succinate-CoA ligase activity or its downstream effects. For example, cells with SUCLA2 knockout can be screened for synthetic lethal interactions or resistance to metabolic stress [3,4]. Such screens have revealed links to glutamine metabolism and redox pathways.
Proteomics and succinylation analysis
Mass spectrometry-based proteomics can quantify succinylation of lysine residues on succinate-CoA ligase subunits and interacting proteins, such as GLS. Immunoprecipitation followed by Western blotting with anti-succinyllysine antibodies provides a targeted approach. These methods help dissect post-translational regulation.
Metabolic flux analysis
Stable isotope tracing with 13C-labeled substrates (e.g., glucose or glutamine) coupled to mass spectrometry can measure flux through the TCA cycle and the contribution of succinate-CoA ligase to succinyl-CoA production [2,6]. This approach is powerful for understanding how the enzyme integrates with other metabolic pathways in health and disease [2,6].
How CRISPR Can Be Used to Study GO:0004774 succinate-CoA ligase activity
Knockout
CRISPR-Cas9 knockout of SUCLA2 or SUCLG1 in cell lines (e.g., HEK293T, HeLa) abolishes succinate-CoA ligase activity, leading to impaired TCA cycle flux, reduced mitochondrial respiration, and mtDNA depletion [1,8]. These models are valuable for studying the metabolic consequences of enzyme loss and for identifying compensatory pathways.
Point Mutation
CRISPR point mutation knock-in can introduce patient-specific missense mutations (e.g., in SUCLG1 or SUCLA2) to model mitochondrial DNA depletion syndromes. Such models allow researchers to dissect the effects of individual mutations on enzyme stability, activity, and cellular phenotypes.
Knock-in
Knock-in of epitope-tagged SUCLA2 or SUCLG1 (e.g., FLAG, HA) enables precise localization, interaction, and proteomic studies. Tagged knock-in models can also be used to rescue knockout phenotypes and confirm that observed effects are due to the specific gene.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of SUCLG2 or SUCLA2 can model gain-of-function effects, such as enhanced stress granule assembly and metastasis in cancer cells. Overexpression models are useful for studying the oncogenic potential of specific isoforms.
How EDITGENE Supports succinate-CoA ligase activity Research
Researchers studying succinate-CoA ligase activity-related genes often need to determine whether a candidate gene is causally involved in mitochondrial metabolism, disease progression, or therapeutic response. Generating precise genetic models is critical to establish causality and to dissect the molecular mechanisms underlying SUCLA2, SUCLG1, and SUCLG2 functions. EDITGENE provides a comprehensive suite of CRISPR-based services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for succinate-CoA ligase activity research.
Frequently Asked Questions About succinate-CoA ligase activity
What is succinate-CoA ligase activity?
Succinate-CoA ligase activity (GO:0004774) is the enzymatic catalysis of the reversible reaction: succinate + CoA + nucleotide triphosphate = succinyl-CoA + nucleotide diphosphate + phosphate. It is a key step in the TCA cycle.
What genes are involved in succinate-CoA ligase activity?
The main genes are SUCLG1 (α-subunit), SUCLA2 (ADP-forming β-subunit), and SUCLG2 (GDP-forming β-subunit). These encode the subunits of the heterodimeric enzyme [1,7].
What diseases are associated with succinate-CoA ligase deficiency?
Mutations in SUCLA2 and SUCLG1 cause mitochondrial DNA depletion syndromes, often presenting as Leigh-like encephalomyopathy. SUCLG2 has been linked to cancer metastasis [1,4,7].
How is succinate-CoA ligase activity regulated?
It is regulated by substrate availability, post-translational modifications such as succinylation and β-hydroxybutyrylation, and interactions with metabolic signaling pathways like AMPK [2,3,6].
What is the role of SUCLA2 in cancer?
SUCLA2 regulates glutaminase (GLS) succinylation and activity, affecting redox balance and tumor cell survival. Its loss can promote metabolic reprogramming.
How can I study succinate-CoA ligase activity in the lab?
Common methods include enzymatic activity assays, CRISPR knockout models, metabolic flux analysis, and proteomics for succinylation [3,5,8].
What is the difference between SUCLA2 and SUCLG2?
SUCLA2 forms the ADP-forming isoform, while SUCLG2 forms the GDP-forming isoform. They share the same α-subunit (SUCLG1) but have distinct nucleotide specificities and tissue distributions.
Can CRISPR be used to model succinate-CoA ligase deficiency?
Yes, CRISPR knockout of SUCLA2 or SUCLG1 in cell lines and animal models recapitulates key features of the disease, such as mtDNA depletion and mitochondrial dysfunction [1,8].
What are the symptoms of SUCLA2-related mitochondrial DNA depletion syndrome?
Symptoms include hypotonia, developmental delay, lactic acidosis, and Leigh-like brain lesions. Onset is typically in infancy or early childhood [1,7].
Is succinate-CoA ligase a target for drug development?
Yes, its role in cancer metabolism and mitochondrial diseases makes it a potential target. However, therapeutic strategies are still in early stages [3,4].
Conclusion
Succinate-CoA ligase activity (GO:0004774) is a central enzymatic function in mitochondrial metabolism, bridging the TCA cycle to nucleotide homeostasis and biosynthetic pathways. Its dysregulation is implicated in severe mitochondrial diseases, cancer, and metabolic disorders, making it a focal point for basic and translational research. Advances in CRISPR-based models and metabolic profiling continue to unravel the complex regulation and tissue-specific roles of SUCLA2, SUCLG1, and SUCLG2. EDITGENE provides the tools and expertise to accelerate these discoveries, from custom knockout and knock-in cell lines to high-throughput screens and bioinformatics support.
References
- 1. Adam MP et al.. 1993. Nuclear Gene-Encoded Leigh Syndrome Spectrum Overview.. PMID: 26425749
- 2. Peng C et al.. 2025. Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK.. Nat Commun 16(1):1738 PMID: 39966410
- 3. 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
- 4. Boese AC et al.. 2023. Succinyl-CoA ligase ADP-forming subunit beta promotes stress granule assembly to regulate redox and drive cancer metastasis.. Proc Natl Acad Sci U S A 120(23):e2217332120 PMID: 37253003
- 5. Luís PB et al.. 2014. Valproyl-CoA inhibits the activity of ATP- and GTP-dependent succinate:CoA ligases.. J Inherit Metab Dis 37(3):353-7 PMID: 24154984
- 6. Han W et al.. 2025. Ketogenic β-hydroxybutyrate regulates β-hydroxybutyrylation of TCA cycle-associated enzymes and attenuates disease-associated pathologies in Alzheimer's mice.. Aging Cell 24(1):e14368 PMID: 39411885
- 7. Demirbas D et al.. 2019. Phenotypic variability in deficiency of the α subunit of succinate-CoA ligase.. JIMD Rep 46(1):63-69 PMID: 31240156
- 8. Lancaster MS et al.. 2024. Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes.. J Cachexia Sarcopenia Muscle 15(6):2729-2742 PMID: 39482887
