GO:0016790 thiolester hydrolase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0016790 thiolester hydrolase activity describes enzymes that hydrolyze thiolester bonds (RCO-SR' + H2O = RCOOH + HSR'), a fundamental reaction in fatty acid metabolism and CoA homeostasis.
• Thioesterases are classified into EC 3.1.2 and include palmitoyl-CoA hydrolase, which regulates cellular acyl-CoA levels and lipid signaling [1,5,8].
• The activity is essential for maintaining the balance of acyl-CoA pools, influencing energy metabolism, membrane synthesis, and protein acylation.
• Dysregulation of thioesterases such as PPT1 is linked to cancer immune evasion and lysosomal storage disorders.
• Palmitoyl-CoA hydrolase activity is modulated by substrate availability, detergents, and dietary fat, indicating tight physiological regulation [2,5,8].
• CRISPR-based knockout, knock-in, and overexpression models enable precise interrogation of thioesterase function in health and disease [3,6].
Description
Thiolester hydrolase activity (GO:0016790) is a molecular function that catalyzes the hydrolysis of thiolester bonds, releasing a carboxylic acid and a thiol. This reaction is central to the metabolism of acetyl-coenzyme A and other acyl-CoA thioesters, which are key intermediates in fatty acid oxidation, lipid biosynthesis, and protein modification. The importance of this activity is underscored by its role in regulating cellular acyl-CoA concentrations, which can otherwise disrupt membrane integrity and signaling [1,5]. Researchers study thiolester hydrolases to understand metabolic disorders, cancer, and immune regulation. The enzyme palmitoyl-CoA hydrolase, for example, is highly sensitive to substrate and detergent environments, reflecting its dynamic regulation in vivo [2,5]. Recent structural and functional studies have expanded the known family of thioesterases, revealing diverse catalytic mechanisms and physiological roles. This article synthesizes current knowledge on GO:0016790, covering its mechanism, key genes, disease associations, and CRISPR-based research strategies.
thiolester hydrolase activity At A Glance
| GO ID | GO:0016790 |
|---|---|
| GO term | thiolester hydrolase activity |
| Ontology | molecular_function |
| Synonym | thiolesterase activity |
| Major function | Hydrolysis of thiolester bonds in acyl-CoA and other thioesters |
| EC number | 3.1.2.- |
| Reaction | RCO-SR' + H2O = RCOOH + HSR' |
| Substrates | Acyl-CoA thioesters, acetyl-CoA, palmitoyl-CoA |
| Products | Free fatty acids, CoA, thiols |
What Is GO:0016790?
Thiolester hydrolase activity (GO:0016790) is defined as the catalysis of the reaction RCO-SR' + H2O = RCOOH + HSR', which is the hydrolysis of a thiolester bond formed between a carboxylic acid and a thiol. This activity is also known as thiolesterase activity and is classified under EC 3.1.2. It includes enzymes such as palmitoyl-CoA hydrolase that cleave acyl-CoA thioesters [1,6].
Why Is thiolester hydrolase activity Important in Cell Biology?
Thiolester hydrolase activity is crucial for maintaining cellular homeostasis by controlling the levels of acyl-CoA thioesters, which are central to energy metabolism and lipid signaling. Dysregulation of this activity can lead to metabolic disorders, cancer, and immune dysfunction. For instance, palmitoyl-CoA hydrolase modulates the availability of palmitoyl-CoA for protein palmitoylation and lipid synthesis, impacting membrane dynamics and signal transduction [1,5]. Moreover, thioesterases like PPT1 regulate STING signaling and immune surveillance, highlighting their therapeutic potential.
• Regulates intracellular acyl-CoA pools, preventing lipotoxicity and metabolic stress.
• Controls fatty acid oxidation and lipid biosynthesis by releasing free fatty acids from CoA.
• Modulates protein palmitoylation and other acylation events by affecting substrate availability.
• Influences immune responses through PPT1-mediated regulation of STING signaling.
• Associated with lysosomal storage disorders such as infantile neuronal ceroid lipofuscinosis.
• Plays a role in cancer cell survival and immune evasion, making it a potential drug target.
• Dietary fat and detergents alter palmitoyl-CoA hydrolase activity, linking nutrition to enzyme function [5,8].
• Bacterial glyoxalases with thioesterase activity contribute to detoxification and virulence.
• Mitochondrial translation and thioesterase activity are linked to cytotoxic T cell killing.
• Provides a model system for studying enzyme kinetics and substrate stabilization.
What Happens During thiolester hydrolase activity?
Substrate Binding and Recognition
In simple terms: The enzyme grabs the thioester molecule, like palmitoyl-CoA, and positions it for cleavage.
Thiolester hydrolases bind their substrates, typically acyl-CoA thioesters, through a conserved catalytic pocket. The enzyme palmitoyl-CoA hydrolase from rat brain shows high affinity for palmitoyl-CoA, and its activity is stabilized by substrate binding [1,2]. The binding involves hydrophobic interactions with the acyl chain and electrostatic interactions with the CoA moiety.
Catalytic Hydrolysis
In simple terms: Water is used to break the bond between the fatty acid and CoA, releasing both parts.
The hydrolysis reaction proceeds via nucleophilic attack of a water molecule on the carbonyl carbon of the thioester bond, forming a tetrahedral intermediate that collapses to release the free carboxylic acid and CoA. This mechanism often involves a catalytic triad (Ser-His-Asp) or a cysteine residue, depending on the enzyme family. For palmitoyl-CoA hydrolase, optimal activity requires specific pH and ionic conditions.
Product Release and Enzyme Turnover
In simple terms: The enzyme lets go of the products and is ready to act again.
After hydrolysis, the free fatty acid and CoA are released from the active site, allowing the enzyme to catalyze another round. The activity of palmitoyl-CoA hydrolase is influenced by the physical state of the substrate, such as micelle formation with detergents. Substrate stabilization studies indicate that the enzyme can be protected from inactivation by its substrate.
Regulation by Cellular Environment
In simple terms: The surrounding conditions, like diet or detergents, can speed up or slow down the enzyme.
Thiolester hydrolase activity is modulated by the lipid environment, detergents, and dietary fat. For example, high-fat diets alter palmitoyl-CoA hydrolase activity in rat liver. Non-ionic detergents such as Triton X-100 affect the enzyme's activity in mixed micelles. These factors highlight the importance of membrane context and substrate presentation.
Key Genes Involved in GO:0016790 thiolester hydrolase activity
The following genes encode enzymes with thiolester hydrolase activity or related thioesterases, each with distinct roles and research relevance.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PPT1 | Palmitoyl-protein thioesterase 1; hydrolyzes palmitoylated proteins | Regulates STING signaling; linked to cancer immunity and neuronal ceroid lipofuscinosis |
| ACOT1 | Acyl-CoA thioesterase 1; hydrolyzes long-chain acyl-CoAs | Regulates lipid metabolism and energy homeostasis |
| ACOT2 | Acyl-CoA thioesterase 2; mitochondrial very-long-chain acyl-CoA thioesterase | Involved in fatty acid oxidation and mitochondrial function |
| ACOT7 | Acyl-CoA thioesterase 7; brain-specific acyl-CoA hydrolase | Modulates neuronal lipid metabolism and signaling |
| ACOT8 | Peroxisomal acyl-CoA thioesterase 8 | Plays a role in peroxisomal fatty acid oxidation |
| ACOT9 | Acyl-CoA thioesterase 9; mitochondrial | Regulates acyl-CoA pools in mitochondria |
| ACOT11 | Acyl-CoA thioesterase 11; also known as THEM1 | Involved in thermogenesis and lipid metabolism |
| ACOT12 | Cytosolic acetyl-CoA hydrolase | Regulates acetyl-CoA levels for lipogenesis |
| ACOT13 | Thioesterase superfamily member 2; hydrolyzes acyl-CoAs | Linked to insulin sensitivity and lipid metabolism |
| BTH | Bacterial thioesterase; involved in glyoxalase system | Detoxifies methylglyoxal; potential antibacterial target |
| PALM | Palmitoyl-CoA hydrolase; hydrolyzes palmitoyl-CoA | Regulates palmitoylation and lipid signaling [1,2] |
| THEM4 | Thioesterase superfamily member 4; acyl-CoA thioesterase | Modulates insulin signaling and cancer |
| THEM5 | Thioesterase superfamily member 5 | Role in lipid metabolism and stress response |
| LYPLA1 | Lysophospholipase 1; also has thioesterase activity | Regulates protein depalmitoylation |
| LYPLA2 | Lysophospholipase 2; thioesterase | Involved in protein palmitoylation cycles |
| PPT2 | Palmitoyl-protein thioesterase 2 | Lysosomal enzyme; may compensate for PPT1 deficiency |
| FASN | Fatty acid synthase; includes thioesterase domain | Produces fatty acids; target for cancer therapy |
| OLAH | Oleoyl-ACP hydrolase; thioesterase | Role in fatty acid synthesis in bacteria and plants |
How Is thiolester hydrolase activity Regulated?
Thiolester hydrolase activity is regulated at multiple levels. Substrate availability and stabilization directly affect enzyme activity, as shown for palmitoyl-CoA hydrolase. Dietary factors, such as high-fat diets, modulate enzyme levels in liver. The physical state of the substrate, including micelle formation with detergents, influences catalytic efficiency. Additionally, post-translational modifications and protein-protein interactions may regulate thioesterases, though specific mechanisms require further study. In immune cells, mitochondrial translation is required for sustained killing by cytotoxic T cells, potentially linking thioesterase activity to metabolic regulation.
thiolester hydrolase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PPT1 | Cancer immune evasion; neuronal ceroid lipofuscinosis | PPT1 knockout cancer cells; patient-derived fibroblasts |
| ACOT1 | Obesity and insulin resistance | ACOT1 knockout mouse; high-fat diet model |
| ACOT2 | Fatty acid oxidation disorders | ACOT2 knockout hepatocytes; mitochondrial stress |
| BTH | Bacterial infections | BTH knockout bacteria; infection model |
| LYPLA1 | Cancer and neurological disorders | LYPLA1 knockdown cell lines; depalmitoylation assays |
Cancer and Immune Evasion
PPT1 acts as a negative regulator of STING signaling in cancer cells, and its inhibition reactivates immune surveillance in cold tumors. This suggests that thiolester hydrolase activity can promote tumor immune evasion, making PPT1 a promising target for cancer immunotherapy.
Neurodegenerative and Lysosomal Storage Disorders
Mutations in PPT1 cause infantile neuronal ceroid lipofuscinosis, a fatal neurodegenerative disease characterized by accumulation of palmitoylated proteins. This highlights the critical role of thiolester hydrolases in neuronal health and lysosomal function.
Metabolic Disorders
Dysregulation of acyl-CoA thioesterases such as ACOT1 and ACOT2 is associated with obesity, insulin resistance, and fatty liver disease. These enzymes control lipid flux and energy balance, and their altered activity contributes to metabolic syndrome.
Infectious Diseases
Bacterial glyoxalase enzymes with thioesterase activity are involved in detoxification of methylglyoxal and contribute to virulence. Targeting these enzymes could provide new antibacterial strategies.
From thiolester hydrolase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of thiolester hydrolase activity affect lipid metabolism? | CRISPR knockout of ACOT1 in HepG2 cells |
| How does a point mutation in the catalytic site alter substrate specificity? | CRISPR point mutation of PPT1 catalytic serine |
| Can a tagged thioesterase be used to track subcellular localization? | Knock-in of GFP-ACOT2 in HeLa cells |
| Does overexpression of PPT1 enhance immune evasion? | Overexpression of PPT1 in melanoma cells |
| What is the role of thioesterase in bacterial detoxification? | Knockout of BTH in E. coli |
| How does dietary fat regulate palmitoyl-CoA hydrolase? | High-fat diet in rats with liver-specific knockout |
How to Study the thiolester hydrolase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Spectrophotometric thioesterase assay | Release of CoA from acyl-CoA | Kinetic analysis of palmitoyl-CoA hydrolase |
| CRISPR knockout | Loss of gene function | Studying ACOT1 in lipid metabolism |
| Overexpression | Gain of function | PPT1 effects on STING signaling |
| Lipidomics | Acyl-CoA and fatty acid levels | Metabolic profiling of thioesterase mutants |
| Western blot | Protein expression and tagging | Validation of knockout/knock-in |
| Immunofluorescence | Subcellular localization | Tagged ACOT2 in mitochondria |
| qRT-PCR | mRNA expression levels | Knockdown efficiency |
| Co-immunoprecipitation | Protein-protein interactions | Identifying thioesterase complexes |
Enzymatic Activity Assays
Thiolester hydrolase activity is commonly measured using spectrophotometric assays that monitor the release of CoA from acyl-CoA substrates. For palmitoyl-CoA hydrolase, activity can be assessed by following the decrease in absorbance at 232 nm due to thioester bond cleavage [1,5]. These assays are sensitive to substrate concentration, detergents, and pH.
Genetic Knockout and Knockdown
CRISPR-Cas9 knockout or siRNA knockdown of specific thioesterase genes allows researchers to study loss-of-function phenotypes. For example, PPT1 knockout in cancer cells increases STING signaling and immune activation. Knockout of ACOT1 in hepatocytes reveals its role in lipid accumulation.
Overexpression and Tagged Fusion Proteins
Overexpression of wild-type or mutant thioesterases, often with GFP or FLAG tags, enables localization and interaction studies. Tagged PPT1 has been used to track lysosomal trafficking. Overexpression of ACOT2 in mitochondria helps assess its impact on fatty acid oxidation.
Lipidomics and Metabolomics
Mass spectrometry-based lipidomics and metabolomics quantify changes in acyl-CoA species and free fatty acids upon modulation of thiolester hydrolase activity. These methods have been applied to study ACOT family enzymes and their impact on cellular lipidomes.
How CRISPR Can Be Used to Study GO:0016790 thiolester hydrolase activity
Knockout
CRISPR-Cas9 knockout of thioesterase genes such as ACOT1 or PPT1 creates cell models to study loss of thiolester hydrolase activity. These models reveal consequences for lipid metabolism, immune signaling, and disease phenotypes [6,7].
Point Mutation
Introducing point mutations in catalytic residues (e.g., serine or cysteine) of thioesterases via CRISPR base editing or HDR allows precise dissection of catalytic mechanism and substrate specificity.
Knock-in
Knock-in of tagged versions (e.g., GFP, FLAG) of thioesterases enables real-time tracking of localization and dynamics. This approach has been used for lysosomal enzymes like PPT1.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of thioesterases such as PPT1 or ACOT2 allows gain-of-function studies, including effects on tumor immune evasion and lipid accumulation [6,7].
How EDITGENE Supports thiolester hydrolase activity Research
Researchers studying thiolester hydrolase activity-related genes often need to determine whether a candidate gene is causally involved in metabolic regulation, immune signaling, or disease progression. EDITGENE provides comprehensive CRISPR-based services to generate precisely engineered cell models, enabling functional validation and mechanistic studies.
Contact EDITGENE today to design your custom CRISPR model for thiolester hydrolase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| PTPRT Knockout HEK293 Cell Line | EDJ-KQ2663 | Human | 11122 | Details Get a Quote |
| PNKD Knockout HEK293 Cell Line | EDJ-KQ3575 | Human | 25953 | Details Get a Quote |
| BAAT Knockout HEK293 Cell Line | EDJ-KQ4118 | Human | 570 | Details Get a Quote |
| ACOT2 Knockout HEK293 Cell Line | EDJ-KQ7229 | Human | 10965 | Details Get a Quote |
| ACOT7 Knockout HEK293 Cell Line | EDJ-KQ7375 | Human | 11332 | Details Get a Quote |
| ACOT11 Knockout HEK293 Cell Line | EDJ-KQ7687 | Human | 26027 | Details Get a Quote |
| ACOT4 Knockout HEK293 Cell Line | EDJ-KQ8169 | Human | 122970 | Details Get a Quote |
| ACOT12 Knockout HEK293 Cell Line | EDJ-KQ9343 | Human | 134526 | Details Get a Quote |
| ACOT1 Knockout HEK293 Cell Line | EDJ-KQ12259 | Human | 641371 | Details Get a Quote |
| ACOT6 Knockout HEK293 Cell Line | EDJ-KQ12260 | Human | 641372 | Details Get a Quote |
| ACOT2 Knockout A-549 Cell Line | EDJ-KQ32201 | Human | 10965 | Details Get a Quote |
| ACOT2 Knockout HeLa Cell Line | EDJ-KQ32202 | Human | 10965 | Details Get a Quote |
| ACOT4 Knockout A-549 Cell Line | EDJ-KQ34078 | Human | 122970 | Details Get a Quote |
| ACOT1 Knockout A-549 Cell Line | EDJ-KQ41052 | Human | 641371 | Details Get a Quote |
| ACOT1 Knockout HeLa Cell Line | EDJ-KQ41053 | Human | 641371 | Details Get a Quote |
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Frequently Asked Questions About thiolester hydrolase activity
What is thiolester hydrolase activity?
Thiolester hydrolase activity (GO:0016790) is the catalysis of the hydrolysis of a thiolester bond, releasing a carboxylic acid and a thiol, such as in acyl-CoA molecules.
What genes are involved in thiolester hydrolase activity?
Key genes include PPT1, ACOT1, ACOT2, ACOT7, and other acyl-CoA thioesterases, as well as bacterial glyoxalases [4,6,7].
What diseases are associated with thiolester hydrolase dysfunction?
Dysfunction is linked to cancer immune evasion, neuronal ceroid lipofuscinosis, metabolic disorders, and bacterial infections [4,6,7].
How is thiolester hydrolase activity measured?
It is commonly measured using spectrophotometric assays that monitor CoA release from acyl-CoA substrates at 232 nm [1,5].
What is the role of PPT1 in cancer?
PPT1 negatively regulates STING signaling, and its inhibition reactivates immune surveillance in cold tumors.
How do CRISPR knockouts help study thioesterases?
CRISPR knockouts of genes like ACOT1 or PPT1 reveal their roles in lipid metabolism, immune signaling, and disease [6,7].
What is the difference between thioesterase and hydrolase?
Thioesterase is a type of hydrolase that specifically cleaves thioester bonds; thiolester hydrolase activity is the GO term for this function.
Can thiolester hydrolase activity be regulated by diet?
Yes, high-fat diets alter palmitoyl-CoA hydrolase activity in rat liver, indicating nutritional regulation.
What are the substrates of thiolester hydrolases?
Common substrates include palmitoyl-CoA, acetyl-CoA, and other acyl-CoA thioesters [1,6].
How does EDITGENE support thiolester hydrolase research?
EDITGENE provides CRISPR knockout, knock-in, point mutation, overexpression, library screening, and bioinformatics services for thioesterase genes [6,7].
Conclusion
Thiolester hydrolase activity (GO:0016790) is a fundamental enzymatic function that regulates acyl-CoA metabolism, lipid signaling, and immune responses. Its dysregulation contributes to cancer, neurodegeneration, and metabolic diseases, making it a compelling target for therapeutic intervention [6,7]. Advances in CRISPR-based models and analytical methods continue to unravel the complex biology of thioesterases, offering new opportunities for drug discovery and precision medicine [3,6].
References
- 1. Knauer TE. 1979. Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain.. Biochem J 179(3):515-23 PMID: 38776
- 2. Knauer TE et al.. 1980. Substrate stabilization of the palmitoyl-coenzyme A hydrolase activity of rat submaxillary gland.. Biochem J 187(1):269-72 PMID: 6105869
- 3. Lisci M et al.. 2021. Mitochondrial translation is required for sustained killing by cytotoxic T cells.. Science 374(6565):eabe9977 PMID: 34648346
- 4. Suttisansanee U et al.. 2011. Bacterial glyoxalase enzymes.. Semin Cell Dev Biol 22(3):285-92 PMID: 21310258
- 5. Berge RK et al.. 1981. Variations in the activity of microsomal palmitoyl-CoA hydrolase in mixed micelle solutions of palmitoyl-CoA and non-ionic detergents of the triton X series.. Biochim Biophys Acta 666(1):25-35 PMID: 6117325
- 6. Swarbrick CMD et al.. 2020. Structure, function, and regulation of thioesterases.. Prog Lipid Res 79:101036 PMID: 32416211
- 7. Chowdhury SR et al.. 2025. PPT1 is a negative regulator of STING signaling in cancer cells and its inhibition reactivates immune surveillance in cold tumors.. Proc Natl Acad Sci U S A 122(39):e2514948122 PMID: 40982692
- 8. Berge RK et al.. 1985. Effects of high fat diets on the activity of palmitoyl-CoA hydrolase in rat liver.. Lipids 20(1):49-52 PMID: 2857471
