GO:0047693 ATP diphosphatase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0047693 ATP diphosphatase activity is a molecular function defined by the QuickGO reaction ATP + H2O = AMP + H+ + diphosphate, also known as ATP pyrophosphatase or ATP diphosphohydrolase activity.
• The best-characterized enzymes carrying this activity are ectonucleoside triphosphate diphosphohydrolases (ENTPDases), especially ENTPD1/CD39, which hydrolyze extracellular ATP to AMP and diphosphate.
• By removing extracellular ATP, ATP diphosphatase activity controls purinergic signaling and limits P2X7-driven inflammation in sepsis and liver injury.
• CD39/ENTPD1 is a marker of antibody-secreting B cells and modulates germinal center and antibody responses during infection.
• Human soluble CD39 displays substrate inhibition in a substrate-specific manner, indicating that ATP diphosphatase activity is tightly regulated by substrate concentration.
• CRISPR knockout, point-mutation, knock-in and overexpression models are essential to dissect the causal roles of ATP diphosphatase enzymes in immunity, inflammation and cancer.
Description
GO:0047693 ATP diphosphatase activity is a molecular function in the Gene Ontology that catalyzes the reaction ATP + H2O = AMP + H+ + diphosphate, meaning it removes two phosphate groups from ATP in a single hydrolytic step to release AMP and pyrophosphate. This activity is synonymous with ATP pyrophosphatase and ATP diphosphohydrolase activity and is best known in the ectonucleoside triphosphate diphosphohydrolase (ENTPDase) family, particularly ENTPD1/CD39. Because extracellular ATP is a potent danger signal, enzymes with ATP diphosphatase activity act as gatekeepers of purinergic signaling and immune homeostasis. Researchers study GO:0047693 to understand how cells terminate ATP-driven inflammatory cascades, how tumors and pathogens evade immune detection, and how nucleotide metabolism shapes cell-cell interactions. The activity is also relevant to infection biology, since CD39 marks antibody-secreting B cells and modulates germinal center responses. In addition, soluble CD39 can show substrate inhibition, revealing that ATP diphosphatase activity is not a simple linear reaction but is tuned by substrate availability. This article integrates the QuickGO definition with verified PubMed literature to provide a research-grade overview of GO:0047693, its genes, mechanisms, disease links and CRISPR-based research methods.
ATP diphosphatase activity At A Glance
| GO ID | GO:0047693 |
|---|---|
| GO term | ATP diphosphatase activity |
| Ontology | molecular_function |
| Synonym | ATP pyrophosphatase activity; ATP diphosphohydrolase; ATP diphosphohydrolase (diphosphate-forming); adenosine triphosphate pyrophosphatase activity |
| Definition | Catalysis of the reaction: ATP + H2O = AMP + H+ + diphosphate |
| Major function | Hydrolysis of ATP to AMP and diphosphate, terminating extracellular ATP signaling |
| Representative enzymes | ENTPD1/CD39 and related ectonucleoside triphosphate diphosphohydrolases |
| Substrate | ATP (adenosine triphosphate) |
| Products | AMP, H+, diphosphate (pyrophosphate) |
| Cofactor requirement | Magnesium-dependent activity has been reported for ATP diphosphohydrolases |
| Biological context | Purinergic signaling, inflammation, immune regulation, cell-cell interactions |
What Is GO:0047693?
In simple terms, ATP diphosphatase activity is the ability of an enzyme to cut ATP into AMP and diphosphate by removing two phosphate groups at once. The QuickGO definition states: Catalysis of the reaction: ATP + H2O = AMP + H+ + diphosphate. This distinguishes it from ATPases that produce ADP and phosphate, and from monophosphatases that act on AMP. The activity is also called ATP pyrophosphatase activity, ATP diphosphohydrolase, ATP diphosphohydrolase (diphosphate-forming), and adenosine triphosphate pyrophosphatase activity. In cells, this function is typically associated with ectoenzymes such as CD39/ENTPD1 that hydrolyze extracellular nucleotides and thereby control purinergic receptor activation.
Why Is ATP diphosphatase activity Important in Cell Biology?
ATP diphosphatase activity is important because it controls the lifetime and concentration of extracellular ATP, a molecule that drives inflammation, thrombosis and immune activation when it accumulates. By converting ATP to AMP, enzymes with this activity simultaneously remove a danger signal and generate a substrate for adenosine production, shifting the local environment from pro-inflammatory to anti-inflammatory. This makes GO:0047693 a central node in diseases such as sepsis, liver injury, thrombosis and cancer, and a target for experimental models that manipulate CD39/ENTPD1.
• Limits P2X7 receptor inflammatory signaling and attenuates sepsis-induced liver injury.
• Regulates thrombo-inflammation through NTPDase1/CD39.
• Controls extracellular ATP and adenosine balance in immune responses.
• Marks antibody-secreting B cells and modulates germinal center and antibody responses during infection.
• Participates in nucleotide metabolism and cell-cell interactions.
• Shows substrate inhibition in human soluble CD39, indicating concentration-dependent regulation.
• Is a potential therapeutic node in liver inflammation and sepsis.
• Can be studied in protozoan parasites such as Herpetomonas muscarum muscarum.
• Provides a mechanism for myeloid cells to restrict T-cell hyperactivation via extracellular vesicles.
• Is a tractable target for CRISPR knockout, knock-in and overexpression studies.
Molecular Mechanism of ATP diphosphatase activity
Substrate recognition and binding of ATP
In simple terms: The enzyme first grabs an ATP molecule from outside the cell.
ATP diphosphatase enzymes such as CD39/ENTPD1 bind extracellular ATP and position it for hydrolysis. This step is the entry point for purinergic signaling control, because removing ATP prevents it from activating P2X7 and other purinergic receptors. The activity is typically measured as the conversion of ATP to AMP and diphosphate, as defined by GO:0047693.
Catalytic hydrolysis of ATP to AMP and diphosphate
In simple terms: The enzyme cuts ATP into AMP and a two-phosphate piece called diphosphate.
The catalytic reaction follows the QuickGO definition: ATP + H2O = AMP + H+ + diphosphate. This is a diphosphohydrolase-type cleavage that removes two phosphate groups in one step, distinguishing it from ATPases that generate ADP. In human soluble CD39, this hydrolysis can display substrate inhibition, meaning high ATP concentrations reduce the reaction rate in a substrate-specific manner.
Magnesium dependence and cofactor requirements
In simple terms: The enzyme often needs magnesium to work properly.
Magnesium-dependent ecto-ATP diphosphohydrolase activity has been reported in Herpetomonas muscarum muscarum, indicating that divalent cations can be required for optimal catalysis. This cofactor dependence is a common feature of NTPDase-family enzymes and should be considered when designing biochemical assays for GO:0047693.
Product release and downstream adenosine generation
In simple terms: After cutting ATP, the products feed into adenosine signaling.
The AMP produced by ATP diphosphatase activity can be further converted to adenosine, which acts as an anti-inflammatory signal. This links GO:0047693 to the Yin and Yang of extracellular ATP and adenosine in immune responses. In sepsis and liver injury models, CD39-mediated ATP removal limits P2X7 inflammatory signaling and attenuates tissue damage.
Regulation by substrate concentration and cellular context
In simple terms: The enzyme's speed depends on how much ATP is around and where it is expressed.
Human soluble CD39 displays substrate inhibition, showing that ATP diphosphatase activity is not a simple linear process but is tuned by substrate levels. In vivo, the activity is also regulated by expression patterns on immune cells, endothelial cells and extracellular vesicles, which together shape cell-cell interactions and inflammation.
Key Genes Involved in GO:0047693 ATP diphosphatase activity
The following genes and proteins are experimentally linked to ATP diphosphatase activity (GO:0047693) or to the purinergic pathways it controls.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ENTPD1 (CD39) | Ectonucleoside triphosphate diphosphohydrolase that hydrolyzes ATP to AMP | Central enzyme for GO:0047693; limits P2X7 signaling in sepsis; regulates thrombo-inflammation |
| P2RX7 | Purinergic receptor activated by extracellular ATP | Downstream target of ATP diphosphatase activity in inflammation |
| ENTPD2 | Related NTPDase family member | Potential alternative enzyme for ATP diphosphatase activity in purinergic signaling |
| ENTPD3 | Related NTPDase family member | Candidate for tissue-specific ATP hydrolysis |
| ENTPD8 | Related NTPDase family member | Candidate for ATP diphosphatase activity in mucosal tissues |
| NT5E (CD73) | Converts AMP to adenosine | Downstream of GO:0047693 in adenosine generation |
| ADORA1 | Adenosine receptor | Mediates anti-inflammatory effects of adenosine produced after ATP hydrolysis |
| ADORA2A | Adenosine receptor | Mediates anti-inflammatory effects of adenosine produced after ATP hydrolysis |
| ADORA2B | Adenosine receptor | Mediates anti-inflammatory effects of adenosine produced after ATP hydrolysis |
| IL1B | Pro-inflammatory cytokine | Readout of P2X7-driven inflammation controlled by CD39 |
| TNF | Pro-inflammatory cytokine | Readout of inflammatory signaling modulated by ATP diphosphatase activity |
| CXCL1 | Chemokine | Inflammation marker in sepsis and liver injury models |
| CD19 | B-cell marker | Used to identify antibody-secreting B cells expressing CD39 |
| PRDM1 (BLIMP1) | Plasma cell transcription factor | Associated with antibody-secreting B cells that express CD39 |
| XBP1 | Plasma cell transcription factor | Associated with antibody-secreting B cells that express CD39 |
| AICDA (AID) | Germinal center enzyme | Germinal center and antibody responses modulated by CD39 |
| HPRT1 | Housekeeping gene | Common control in expression studies of purinergic genes |
| ACTB | Housekeeping gene | Common control in expression studies of purinergic genes |
How Is ATP diphosphatase activity Regulated?
ATP diphosphatase activity is regulated at multiple levels. At the enzyme level, human soluble CD39 displays substrate inhibition, meaning that high ATP concentrations can reduce the reaction rate in a substrate-specific manner. At the expression level, CD39/ENTPD1 is induced on immune cells, endothelial cells and extracellular vesicles, which changes the local capacity for ATP hydrolysis. At the signaling level, the balance between extracellular ATP and adenosine determines whether purinergic signaling is pro-inflammatory or anti-inflammatory, as reviewed in the Yin and Yang concept. In disease contexts such as sepsis, this regulation is critical because loss of ATP diphosphatase activity amplifies P2X7-driven inflammation and tissue injury. Thrombo-inflammation studies further show that NTPDase1/CD39 is a key regulator of platelet-leukocyte interactions and vascular inflammation.
ATP diphosphatase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ENTPD1 (CD39) | Sepsis-induced liver injury | Knockout mouse or CRISPR KO hepatocyte/immune cell lines |
| ENTPD1 (CD39) | Thrombo-inflammation | Platelet-specific KO or knock-in models |
| ENTPD1 (CD39) | Infection and antibody responses | B-cell-specific KO or reporter knock-in |
| P2RX7 | P2X7-driven inflammation | Point-mutation or KO models to block ATP sensing |
| NT5E (CD73) | Adenosine-mediated immunosuppression | Overexpression or KO models to shift ATP/adenosine balance |
Sepsis and liver injury
CD39-mediated ATP diphosphatase activity limits P2X7 receptor inflammatory signaling and attenuates sepsis-induced liver injury, indicating that loss of this activity worsens tissue damage. In related models, engaging natural regulatory myeloid cells restricts T-cell hyperactivation-induced liver inflammation via extracellular vesicle-mediated purine metabolism regulation.
Thrombo-inflammation and vascular disease
NTPDase1/CD39 is a central regulator of thrombo-inflammation, where it controls extracellular ATP levels and platelet-leukocyte interactions. This positions GO:0047693 as a potential target for modulating thrombosis and vascular inflammation.
Infection and antibody responses
CD39 is an antibody-secreting B-cell marker that modulates germinal center and antibody responses during infection, linking ATP diphosphatase activity to humoral immunity. This suggests that pathogens or vaccines may be influenced by the level of CD39 activity on B cells.
Cancer and immune evasion
Because ATP diphosphatase activity removes a danger signal and promotes adenosine generation, it can contribute to an immunosuppressive microenvironment in tumors. The balance between extracellular ATP and adenosine is therefore a key consideration in cancer immunology.
From ATP diphosphatase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of ENTPD1/CD39 increase ATP-driven inflammation? | CRISPR knockout of ENTPD1 in immune or liver cell lines |
| Does a specific catalytic residue mediate ATP diphosphatase activity? | Point mutation of predicted catalytic residues in ENTPD1 |
| Can a tagged CD39 be used to track localization? | Knock-in of an epitope tag at the endogenous ENTPD1 locus |
| Does overexpression of CD39 reduce P2X7 signaling? | Overexpression of ENTPD1 in macrophages or hepatocytes |
| Does substrate inhibition alter ATP hydrolysis in cells? | Knock-in of soluble CD39 variants and biochemical assays |
| Can extracellular vesicles transfer ATP diphosphatase activity? | Overexpression or KO in EV-producing myeloid cells |
How to Study the ATP diphosphatase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Malachite green or coupled enzyme assay | Phosphate or diphosphate release from ATP | Biochemical measurement of ATP diphosphatase activity |
| HPLC or LC-MS nucleotide quantification | ATP, ADP, AMP and adenosine levels | Purinergic balance in cells and tissues |
| ELISA or cytokine bead array | IL1B, TNF and other inflammatory cytokines | P2X7-driven inflammation readouts |
| Flow cytometry | CD39 surface expression on B cells and immune subsets | Immune phenotyping during infection |
| Immunohistochemistry | Tissue localization of CD39/ENTPD1 | Liver injury and thrombo-inflammation models |
| Extracellular vesicle isolation and co-culture | Transfer of purine-metabolizing activity | Myeloid-T cell interaction studies |
| CRISPR knockout screening | Gene requirement for ATP diphosphatase activity | Discovery of novel regulators |
| Reporter knock-in imaging | Real-time localization of CD39 | Live-cell tracking of enzyme trafficking |
Biochemical ATP hydrolysis assays
ATP diphosphatase activity is typically measured by incubating enzyme samples with ATP and detecting the formation of AMP and diphosphate, as defined by GO:0047693. These assays can reveal substrate inhibition, as shown for human soluble CD39.
Purinergic signaling and inflammation readouts
Downstream effects of ATP diphosphatase activity can be measured by monitoring P2X7-dependent inflammatory cytokines such as IL1B and TNF in sepsis or liver injury models. Extracellular ATP and adenosine levels can also be quantified to assess the balance described in the Yin and Yang concept.
Immune cell phenotyping and germinal center analysis
Flow cytometry and immunohistology can identify CD39-positive antibody-secreting B cells and assess germinal center responses during infection. These methods link ATP diphosphatase activity to humoral immunity.
Extracellular vesicle and cell-cell interaction studies
Because purine metabolism can be transferred via extracellular vesicles, EV isolation and co-culture experiments are used to test whether ATP diphosphatase activity modulates T-cell hyperactivation and liver inflammation.
How CRISPR Can Be Used to Study GO:0047693 ATP diphosphatase activity
Knockout
CRISPR knockout of ENTPD1/CD39 is used to test whether loss of ATP diphosphatase activity increases extracellular ATP and amplifies P2X7-driven inflammation in sepsis and liver injury models. Knockout of related NTPDase genes can reveal redundancy or tissue-specific roles in purinergic signaling.
Point Mutation
Point mutation of predicted catalytic residues in CD39 can distinguish ATP diphosphatase activity from other functions and test the mechanism of substrate inhibition observed in human soluble CD39. Such mutants are valuable for separating catalytic activity from protein-protein interactions.
Knock-in
Knock-in of epitope tags or fluorescent reporters at the endogenous ENTPD1 locus allows tracking of CD39 localization and expression without overexpression artifacts. Knock-in of disease-associated variants can model how altered ATP diphosphatase activity contributes to thrombo-inflammation.
Overexpression
Overexpression of CD39 or other NTPDases is used to test whether increasing ATP diphosphatase activity is sufficient to reduce inflammatory signaling and tissue damage. Overexpression in extracellular vesicle-producing cells can also test whether purine metabolism can be transferred to restrict T-cell hyperactivation.
How EDITGENE Supports ATP diphosphatase activity Research
Researchers studying ATP diphosphatase activity-related genes often need to determine whether a candidate gene is causally involved in ATP hydrolysis, purinergic signaling and inflammation, or whether it is merely a bystander. This requires precise, reproducible CRISPR models that can knockout, mutate, tag or overexpress the gene of interest in relevant cell types.
Contact EDITGENE today to design your custom CRISPR model for ATP diphosphatase activity research.
Frequently Asked Questions About ATP diphosphatase activity
What is ATP diphosphatase activity?
ATP diphosphatase activity (GO:0047693) is a molecular function that catalyzes the reaction ATP + H2O = AMP + H+ + diphosphate, removing two phosphate groups from ATP to produce AMP and diphosphate.
What genes are involved in ATP diphosphatase activity?
The best-characterized genes are ENTPD1/CD39 and related ectonucleoside triphosphate diphosphohydrolases, which hydrolyze extracellular ATP and regulate purinergic signaling.
What is the difference between ATP diphosphatase and ATPase?
ATP diphosphatase activity removes two phosphate groups to yield AMP and diphosphate, whereas typical ATPases remove one phosphate to yield ADP and phosphate, as defined by GO:0047693.
How is ATP diphosphatase activity measured?
It is commonly measured by incubating samples with ATP and detecting AMP and diphosphate formation using biochemical assays, HPLC or LC-MS.
Why is CD39 important for ATP diphosphatase activity?
CD39 (ENTPD1) is a major enzyme with ATP diphosphatase activity that limits P2X7 inflammatory signaling and attenuates sepsis-induced liver injury.
Does ATP diphosphatase activity require magnesium?
Magnesium-dependent ecto-ATP diphosphohydrolase activity has been reported, indicating that divalent cations can be required for optimal catalysis.
What diseases are linked to ATP diphosphatase activity?
It has been linked to sepsis, liver injury, thrombo-inflammation, infection and cancer through its role in controlling extracellular ATP and adenosine.
Can ATP diphosphatase activity be inhibited by substrate?
Yes, human soluble CD39 displays substrate inhibition in a substrate-specific manner, meaning high ATP concentrations can reduce the reaction rate.
How do CRISPR models help study ATP diphosphatase activity?
CRISPR knockout, point mutation, knock-in and overexpression models allow researchers to test causal roles of ENTPD1/CD39 and related genes in ATP hydrolysis and inflammation.
What is the GO ID for ATP diphosphatase activity?
The GO ID is GO:0047693, and the official name is ATP diphosphatase activity.
Conclusion
GO:0047693 ATP diphosphatase activity is a molecular function that converts ATP to AMP and diphosphate, and it is central to purinergic signaling, inflammation and immune regulation. The best-studied enzyme carrying this activity is CD39/ENTPD1, which limits P2X7-driven inflammation, modulates thrombo-inflammation and marks antibody-secreting B cells. Because the activity is regulated by substrate concentration and cellular context, precise CRISPR models are essential to dissect its causal roles in disease. Researchers can now use knockout, point-mutation, knock-in and overexpression strategies to advance this field.
References
- 1. Savio LEB et al.. 2017. CD39 limits P2X7 receptor inflammatory signaling and attenuates sepsis-induced liver injury.. J Hepatol 67(4):716-726 PMID: 28554875
- 2. Morello S et al.. 2021. Thrombo-Inflammation: A Focus on NTPDase1/CD39.. Cells 10(9) PMID: 34571872
- 3. Yang F et al.. 2024. Engaging natural regulatory myeloid cells to restrict T-cell hyperactivation-induced liver inflammation via extracellular vesicle-mediated purine metabolism regulation.. Theranostics 14(12):4874-4893 PMID: 39239508
- 4. Almada L et al.. 2025. CD39 is an antibody-secreting B-cell marker that modulates germinal center and antibody responses during infection.. Front Immunol 16:1547929 PMID: 41190065
- 5. Alves-Ferreira M et al.. 2003. Magnesium-dependent ecto-ATP diphosphohydrolase activity in Herpetomonas muscarum muscarum.. Curr Microbiol 47(4):265-71 PMID: 14629005
- 6. Eltzschig HK et al.. 2006. Nucleotide metabolism and cell-cell interactions.. Methods Mol Biol 341:73-87 PMID: 16799190
- 7. Faas MM et al.. 2017. Extracellular ATP and adenosine: The Yin and Yang in immune responses?. Mol Aspects Med 55:9-19 PMID: 28093236
- 8. Vadlamani VMK et al.. 2023. Human soluble CD39 displays substrate inhibition in a substrate-specific manner.. Sci Rep 13(1):8958 PMID: 37268726
