GO:0047710 bis(5'-adenosyl)-triphosphatase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0047710 describes bis(5'-adenosyl)-triphosphatase activity, the enzymatic hydrolysis of P1,P3-bis(5'-adenosyl) triphosphate (AP3A) to ADP and AMP.
• The reaction is a dinucleoside polyphosphate hydrolase step that removes the triphosphate bridge of AP3A and releases two mononucleotides.
• FHIT is the best-characterized human enzyme with this activity, and its loss is linked to common fragile site FRA3B instability and cancer [6,8].
• The enzymatic activity is conserved in prokaryotes and eukaryotes, and bacterial 5'-nucleotidase can also hydrolyze bis(5'-nucleosidyl) polyphosphates.
• Loss of FHIT and related AP3A hydrolase activity is associated with pancreatic cancer and other malignancies [2,3].
• Small-molecule inhibitors of FHIT have been developed, showing that this activity is chemically tractable for experimental perturbation.
Description
GO:0047710, bis(5'-adenosyl)-triphosphatase activity, is a molecular function that catalyzes the hydrolysis of P1,P3-bis(5'-adenosyl) triphosphate (AP3A) into ADP, AMP, and two protons. This activity belongs to the dinucleoside polyphosphate hydrolase family and is often referred to as AP3A hydrolase or AP3Aase. The reaction is chemically simple but biologically significant because AP3A and related dinucleoside polyphosphates can accumulate under stress and influence nucleotide signaling. Researchers study this activity to understand how cells maintain dinucleotide homeostasis and how its loss contributes to genome instability [6,8]. The human enzyme most closely associated with this activity is FHIT, a tumor suppressor encoded at the FRA3B common fragile site [6,8]. FHIT loss is one of the most common alterations in human cancer, making GO:0047710 a relevant function for cancer biology and genome maintenance [2,3,6].
bis(5'-adenosyl)-triphosphatase activity At A Glance
| GO ID | GO:0047710 |
|---|---|
| GO term | bis(5'-adenosyl)-triphosphatase activity |
| Ontology | molecular_function |
| Synonym | AP3Aase activity; AP3A hydrolase activity; diadenosine 5',5'''-P1,P3-triphosphate hydrolase activity |
| Major function | Hydrolysis of P1,P3-bis(5'-adenosyl) triphosphate to ADP and AMP |
| Reaction | P1,P3-bis(5'-adenosyl) triphosphate + H2O = ADP + AMP + 2 H+ |
| Substrate | P1,P3-bis(5'-adenosyl) triphosphate (AP3A) |
| Products | ADP, AMP, and two protons |
| Related activity | Dinucleoside polyphosphate hydrolase; 5'-nucleotidase can also hydrolyze bis(5'-nucleosidyl) polyphosphates |
What Is GO:0047710?
In my own words, GO:0047710 describes an enzyme that uses water to split AP3A, a molecule made of two adenosine nucleotides joined by a triphosphate bridge, into ADP and AMP while releasing two protons. This is a hydrolase reaction that breaks the phosphoanhydride bond between the two adenosines, effectively dismantling the dinucleoside triphosphate. The activity is also known as AP3A hydrolase, AP3Aase, or diadenosine 5',5'''-P1,P3-triphosphate hydrolase.
Why Is bis(5'-adenosyl)-triphosphatase activity Important in Cell Biology?
GO:0047710 is important because it controls the cellular levels of AP3A, a dinucleoside polyphosphate that can influence nucleotide signaling and genome stability [5,6]. The human enzyme FHIT, which carries this activity, is a tumor suppressor located at the FRA3B fragile site, and its loss is among the earliest and most frequent events in many cancers [6,8]. Understanding this activity helps explain how cells prevent the accumulation of potentially harmful dinucleoside polyphosphates and how fragile site instability contributes to cancer development [2,3,6].
• Loss of FHIT AP3A hydrolase activity is linked to pancreatic cancer and other malignancies [2,3].
• FHIT is located at FRA3B, a common fragile site that is frequently deleted in cancer.
• AP3A hydrolase activity helps maintain dinucleoside polyphosphate homeostasis.
• Bacterial 5'-nucleotidase can hydrolyze bis(5'-nucleosidyl) polyphosphates, showing broad conservation.
• Small-molecule inhibitors of FHIT provide tools to probe this activity in cells.
• The activity is relevant to genome maintenance and DNA damage response pathways [6,8].
• FHIT loss may serve as a biomarker for environmental carcinogen exposure and cancer risk [2,3].
• Studying GO:0047710 can reveal new therapeutic targets in cancers with FHIT deficiency [7,8].
What Happens During bis(5'-adenosyl)-triphosphatase activity?
Substrate recognition and binding
In simple terms: The enzyme grabs AP3A, a molecule made of two adenosines linked by three phosphates.
The enzyme binds P1,P3-bis(5'-adenosyl) triphosphate (AP3A) in its active site. AP3A is a dinucleoside polyphosphate composed of two adenosine moieties connected by a triphosphate bridge. The binding step positions the triphosphate bridge for nucleophilic attack by water. This substrate specificity distinguishes GO:0047710 from other hydrolases that act on mononucleotides or longer dinucleoside polyphosphates.
Hydrolytic cleavage of the triphosphate bridge
In simple terms: Water splits the phosphate bridge, breaking AP3A into two smaller pieces.
A water molecule attacks the phosphoanhydride bond of AP3A, leading to cleavage of the triphosphate bridge. The reaction yields ADP and AMP as the principal nucleotide products, along with two protons. This hydrolysis is characteristic of bis(5'-adenosyl)-triphosphatase activity and is distinct from simple phosphomonoesterase reactions.
Product release and dinucleotide homeostasis
In simple terms: The enzyme releases ADP and AMP, helping the cell keep dinucleotide levels under control.
After cleavage, ADP and AMP are released from the active site. The removal of AP3A by this activity prevents the accumulation of dinucleoside polyphosphates, which can otherwise interfere with nucleotide signaling and genome stability [5,6]. In human cells, FHIT is the primary enzyme associated with this activity, and its loss leads to elevated AP3A levels and increased fragile site instability [6,8].
Conservation and alternative enzymes
In simple terms: Other enzymes, even in bacteria, can perform a similar reaction.
The ability to hydrolyze bis(5'-nucleosidyl) polyphosphates is not limited to FHIT. Escherichia coli 5'-nucleotidase can also hydrolyze bis(5'-nucleosidyl) polyphosphates, demonstrating that this activity is evolutionarily conserved. This conservation suggests that dinucleoside polyphosphate hydrolysis is an ancient and important cellular function.
Key Genes Involved in GO:0047710 bis(5'-adenosyl)-triphosphatase activity
The following genes and proteins are directly or indirectly associated with bis(5'-adenosyl)-triphosphatase activity (GO:0047710) and its biological context.
| Gene | Major Role | Research Relevance |
|---|---|---|
| FHIT | Human bis(5'-adenosyl)-triphosphatase (AP3A hydrolase); tumor suppressor at FRA3B | Frequent loss in cancers; linked to fragile site instability [6,8] |
| HIT1 | Yeast homolog of FHIT; dinucleoside polyphosphate hydrolase | Model for studying AP3A hydrolase function in eukaryotes |
| NUDT1 | Nudix hydrolase family member; can hydrolyze dinucleoside polyphosphates | Potential alternative enzyme for AP3A metabolism |
| NUDT2 | Nudix hydrolase; hydrolyzes diadenosine polyphosphates | Related dinucleoside polyphosphate hydrolase |
| NUDT3 | Nudix hydrolase; acts on dinucleoside polyphosphates | May contribute to AP3A turnover |
| NUDT4 | Nudix hydrolase; dinucleoside polyphosphate hydrolase | Candidate modifier of AP3A levels |
| NUDT5 | Nudix hydrolase; hydrolyzes ADP-ribose and dinucleoside polyphosphates | Broad substrate specificity |
| NUDT9 | Nudix hydrolase; ADP-ribose pyrophosphatase | Related nucleotide hydrolase |
| NUDT12 | Nudix hydrolase; NADH pyrophosphatase | Indirect role in nucleotide homeostasis |
| NUDT14 | Nudix hydrolase; UDP-glucose pyrophosphatase | Related nucleotide sugar hydrolase |
| NUDT16 | Nudix hydrolase; decapping enzyme | RNA processing and nucleotide metabolism |
| NUDT18 | Nudix hydrolase; ADP-ribose pyrophosphatase | Related to dinucleotide metabolism |
| NUDT19 | Nudix hydrolase; CoA diphosphatase | Peroxisomal nucleotide metabolism |
| NUDT21 | Nudix hydrolase; mRNA cleavage factor | RNA 3' end processing |
| NUDT22 | Nudix hydrolase; UDP-glucose pyrophosphatase | Nucleotide sugar metabolism |
| 5'-nucleotidase (bacterial) | Hydrolyzes bis(5'-nucleosidyl) polyphosphates in E. coli | Prokaryotic model for AP3A hydrolysis |
| CTNNB1 | Beta-catenin; interacts with FHIT in Wnt signaling | Context for FHIT-related pathways |
| AP3A | Substrate of the reaction | Direct biochemical readout of enzyme activity |
How Is bis(5'-adenosyl)-triphosphatase activity Regulated?
The regulation of bis(5'-adenosyl)-triphosphatase activity is not fully understood, but several lines of evidence point to transcriptional and post-transcriptional control of FHIT. FHIT expression is frequently reduced by deletions and epigenetic silencing at FRA3B, which indirectly lowers AP3A hydrolase activity [6,8]. In addition, FHIT has been reported to interact with beta-catenin, linking this activity to Wnt signaling regulation. Small-molecule inhibitors of FHIT can acutely modulate its enzymatic function, providing a chemical approach to regulate this activity. However, direct allosteric or post-translational regulation of the enzyme remains an area of active investigation.
bis(5'-adenosyl)-triphosphatase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| FHIT | Pancreatic cancer, fragile site instability | FHIT knockout pancreatic cell lines; AP3A hydrolysis assay [2,6] |
| FHIT | Lung cancer, environmental carcinogenesis | FHIT knockout lung epithelial cells; carcinogen exposure [3,6] |
| FHIT | Wnt signaling dysregulation | FHIT knockout with beta-catenin reporter |
| FHIT | Genome instability and DNA damage | FHIT knockout cells with replication stress inducers [6,8] |
| FHIT | Therapeutic target | FHIT inhibitor treatment in cancer cell lines |
Cancer and fragile site instability
Loss of FHIT, the main human enzyme with bis(5'-adenosyl)-triphosphatase activity, is one of the most common genetic alterations in human cancer [6,8]. FHIT spans the FRA3B common fragile site, which is prone to breakage and deletion under replication stress. Reduced AP3A hydrolase activity leads to accumulation of AP3A and contributes to genome instability, promoting tumor development [6,8]. Pancreatic cancer frequently shows FHIT abnormalities, and other cancers also exhibit FHIT loss [2,3].
Pancreatic cancer
Genetic abnormalities in pancreatic cancer include frequent loss of FHIT at 3p14.2. This loss reduces bis(5'-adenosyl)-triphosphatase activity and may contribute to the aggressive phenotype of pancreatic tumors [2,3]. Studies have suggested that FHIT loss can be an early event in pancreatic carcinogenesis.
Environmental carcinogenesis
FHIT alterations have been associated with exposure to environmental carcinogens such as tobacco smoke. Because FHIT is a fragile site gene, it is particularly susceptible to damage by carcinogens, and loss of its AP3A hydrolase activity may serve as a biomarker for exposure and cancer risk [3,6].
From bis(5'-adenosyl)-triphosphatase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of FHIT increase AP3A levels? | FHIT knockout cell line (e.g., HCT116 or HeLa) |
| Does a point mutation in the catalytic site abolish AP3A hydrolase activity? | FHIT point-mutant knock-in cell line |
| Can tagged FHIT be used to monitor localization? | Knock-in of FLAG- or GFP-FHIT |
| Does FHIT overexpression suppress tumor growth? | FHIT overexpression in cancer cell lines |
| Which genes are required for AP3A homeostasis? | CRISPR library screening for AP3A levels |
| Does FHIT loss alter Wnt signaling? | FHIT knockout with beta-catenin reporter |
How to Study the bis(5'-adenosyl)-triphosphatase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| AP3A hydrolysis assay | Enzymatic conversion of AP3A to ADP and AMP | Kinetics and inhibitor testing [5,7] |
| CRISPR knockout | Loss-of-function phenotype | AP3A accumulation and genome instability |
| CRISPR point mutation | Catalytic residue requirement | Active-site mutant analysis |
| Knock-in tagging | Protein localization and interactions | FHIT imaging and immunoprecipitation |
| Overexpression | Gain-of-function effects | Tumor suppression assays |
| RNA-seq | Transcriptional changes | Pathway analysis in FHIT-null cells |
| Proteomics | Protein interaction networks | FHIT interactome |
| Small-molecule screening | Inhibitor discovery | Chemical probes for FHIT |
Biochemical AP3A hydrolysis assay
The most direct way to measure bis(5'-adenosyl)-triphosphatase activity is an in vitro assay using AP3A as substrate and detecting ADP and AMP production by HPLC or coupled enzyme reactions. This method can be used with purified recombinant FHIT or cell lysates to quantify enzyme kinetics and inhibitor effects.
CRISPR knockout and phenotypic analysis
CRISPR-Cas9 knockout of FHIT or related hydrolases allows researchers to test how loss of GO:0047710 activity affects AP3A levels, cell proliferation, and genome stability [6,8]. Knockout cells can be challenged with replication stress agents to reveal fragile site phenotypes.
Small-molecule inhibitor studies
Small-molecule inhibitors of FHIT have been developed and can be used to acutely inhibit AP3A hydrolase activity in cells. These compounds enable dose-dependent studies of AP3A accumulation and downstream signaling without genetic manipulation.
Omics and bioinformatics
Transcriptomic and proteomic profiling of FHIT-deficient cells can reveal pathways altered by loss of AP3A hydrolase activity [6,8]. Bioinformatics analysis of cancer genomes can identify FHIT deletions and mutations that impair enzymatic function [2,6].
How CRISPR Can Be Used to Study GO:0047710 bis(5'-adenosyl)-triphosphatase activity
Knockout
CRISPR knockout of FHIT or other AP3A hydrolases eliminates bis(5'-adenosyl)-triphosphatase activity, allowing researchers to study the consequences of AP3A accumulation [6,8]. Knockout cell lines can be used to test sensitivity to DNA-damaging agents and to identify synthetic lethal interactions.
Point Mutation
Point mutations in the catalytic site of FHIT can be introduced by CRISPR to dissect which residues are essential for AP3A hydrolysis. Such mutants help distinguish enzymatic activity from non-enzymatic functions of FHIT.
Knock-in
Knock-in of epitope tags or fluorescent proteins at the endogenous FHIT locus enables real-time tracking of the enzyme and its interactions. This approach preserves native regulation and can be combined with live-cell imaging.
Overexpression
CRISPR activation or cDNA overexpression can increase FHIT levels to test whether enhanced AP3A hydrolase activity suppresses tumor phenotypes. Overexpression models are useful for gain-of-function studies and for validating inhibitor specificity.
How EDITGENE Supports bis(5'-adenosyl)-triphosphatase activity Research
Researchers studying bis(5'-adenosyl)-triphosphatase activity-related genes often need to determine whether a candidate gene is causally involved in AP3A metabolism, genome stability, or cancer phenotypes. EDITGENE provides a comprehensive suite of CRISPR services to enable these investigations.
Contact EDITGENE today to design your custom CRISPR model for bis(5'-adenosyl)-triphosphatase activity research.
Frequently Asked Questions About bis(5'-adenosyl)-triphosphatase activity
What is bis(5'-adenosyl)-triphosphatase activity?
It is the enzymatic activity defined by GO:0047710 that hydrolyzes P1,P3-bis(5'-adenosyl) triphosphate (AP3A) to ADP and AMP.
What genes are involved in bis(5'-adenosyl)-triphosphatase activity?
The main human gene is FHIT, which encodes a tumor suppressor with AP3A hydrolase activity [6,8]. Other Nudix hydrolases may also contribute.
What is the reaction catalyzed by GO:0047710?
The reaction is P1,P3-bis(5'-adenosyl) triphosphate + H2O = ADP + AMP + 2 H+.
Why is FHIT important for bis(5'-adenosyl)-triphosphatase activity?
FHIT is the best-characterized human enzyme with this activity, and its loss is linked to fragile site instability and cancer [6,8].
How is bis(5'-adenosyl)-triphosphatase activity measured?
It is typically measured using an in vitro assay with AP3A as substrate and detection of ADP and AMP by HPLC or coupled enzymes [5,7].
What diseases are associated with loss of bis(5'-adenosyl)-triphosphatase activity?
Loss of FHIT activity is associated with pancreatic cancer, lung cancer, and other malignancies [2,3,6].
Can CRISPR be used to study bis(5'-adenosyl)-triphosphatase activity?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models can be used to dissect the function of FHIT and related genes [6,8].
What are the synonyms for bis(5'-adenosyl)-triphosphatase activity?
Synonyms include AP3Aase activity, AP3A hydrolase activity, and diadenosine 5',5'''-P1,P3-triphosphate hydrolase activity.
Is bis(5'-adenosyl)-triphosphatase activity conserved in bacteria?
Yes, Escherichia coli 5'-nucleotidase can hydrolyze bis(5'-nucleosidyl) polyphosphates, indicating conservation.
What are small-molecule inhibitors of FHIT?
Small-molecule inhibitors of FHIT have been developed as chemical tools to probe AP3A hydrolase activity.
Conclusion
GO:0047710, bis(5'-adenosyl)-triphosphatase activity, is a conserved enzymatic function that controls the levels of the dinucleoside polyphosphate AP3A. The human enzyme FHIT is the most studied protein with this activity, and its loss is a frequent event in cancer that contributes to genome instability [6,8]. Understanding this activity provides insights into nucleotide metabolism, fragile site biology, and tumor suppression [2,3]. CRISPR-based models and biochemical assays are powerful tools for further dissecting the role of this activity in health and disease [6,7].
References
- 1. Huber O et al.. 2008. Beta-catenin takes a HIT.. Cell Cycle 7(10):1326-31 PMID: 18596417
- 2. Moore PS et al.. 2003. Genetic abnormalities in pancreatic cancer.. Mol Cancer 2:7 PMID: 12537585
- 3. Yoshida T et al.. 1997. Oncology.. JAMA 277(23):1880-1 PMID: 9185814
- 5. Ruiz A et al.. 1989. Hydrolysis of bis(5'-nucleosidyl) polyphosphates by Escherichia coli 5'-nucleotidase.. J Bacteriol 171(12):6703-9 PMID: 2556371
- 6. Saldivar JC et al.. 2019. Mechanisms shaping the mutational landscape of the FRA3B/FHIT-deficient cancer genome.. Genes Chromosomes Cancer 58(5):317-323 PMID: 30242938
- 7. Lange S et al.. 2017. Small-Molecule Inhibitors of the Tumor Suppressor Fhit.. Chembiochem 18(17):1707-1711 PMID: 28643453
- 8. Huebner K et al.. 2011. Hits, Fhits and Nits: beyond enzymatic function.. Adv Enzyme Regul 51(1):208-17 PMID: 21035495
