GO:0004638 phosphoribosylaminoimidazole carboxylase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004638 describes the enzymatic decarboxylation of 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate (CAIR) to 5-amino-1-(5-phospho-D-ribosyl)imidazole (AIR) and CO2, a step in de novo purine biosynthesis.
• In humans, this activity is carried by the N-terminal domain of the bifunctional enzyme PAICS, which also catalyzes the subsequent SAICAR synthetase step.
• PAICS is overexpressed in multiple cancers, including non-small cell lung cancer, gastric cancer, and breast cancer, and is considered an emerging therapeutic target.
• The enzyme functions as a homooctamer and exhibits substrate channeling between its carboxylase and synthetase domains.
• Loss of phosphoribosylaminoimidazole carboxylase activity causes purine auxotrophy, as demonstrated in Chinese hamster ovary Ade-D mutants.
• CRISPR knockout, point mutation, and overexpression models are essential for dissecting PAICS function in purine metabolism and cancer.
Description
Phosphoribosylaminoimidazole carboxylase activity (GO:0004638) is a molecular function that catalyzes the conversion of 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate (CAIR) to 5-amino-1-(5-phospho-D-ribosyl)imidazole (AIR) with the release of carbon dioxide. This reaction constitutes the sixth step of the de novo purine biosynthesis pathway, a fundamental metabolic route for the production of purine nucleotides. In humans, the enzyme responsible for this activity is PAICS (phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase), a bifunctional protein that also catalyzes the subsequent SAICAR synthetase reaction. The carboxylase domain of PAICS is classified as a class II PurE enzyme, and its activity is essential for cellular proliferation because purines are required for DNA, RNA, ATP, GTP, and cofactor synthesis. Research on GO:0004638 has gained prominence due to the recognition that cancer cells often reprogram purine metabolism to support rapid growth. PAICS is overexpressed in several malignancies, and its knockdown or inhibition suppresses malignant proliferation, making it a candidate for targeted therapy. Structural and biochemical studies have revealed detailed mechanisms of substrate binding and product release, including evidence for substrate channeling between the carboxylase and synthetase domains. Additionally, classical genetic studies in Chinese hamster ovary cells established that loss of this activity leads to purine auxotrophy, linking the enzyme to the Ade-D locus on human chromosome 4. For researchers, understanding GO:0004638 is critical for investigating purine-related diseases, developing anticancer drugs, and interpreting metabolic flux in normal and pathological tissues. The availability of CRISPR-based tools now enables precise manipulation of PAICS and related genes to test causal roles in disease models.
phosphoribosylaminoimidazole carboxylase activity At A Glance
| GO ID | GO:0004638 |
|---|---|
| GO term | phosphoribosylaminoimidazole carboxylase activity |
| Ontology | molecular_function |
| Synonym | AIR carboxylase activity; class II PurE; ADE2; 5-phosphoribosyl-5-aminoimidazole carboxylase activity |
| Major function | Catalyzes the decarboxylation of CAIR to AIR and CO2 in de novo purine biosynthesis |
| EC number | 4.1.1.21 |
| Reaction direction | Irreversible decarboxylation |
| Cofactors | None required; class II PurE enzymes are metal-independent |
| Subcellular location | Cytoplasm (in humans, PAICS is cytosolic) |
What Is GO:0004638?
Phosphoribosylaminoimidazole carboxylase activity (GO:0004638) is defined as the catalysis of the reaction: 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate + 2 H+ = 5-amino-1-(5-phospho-D-ribosyl)imidazole + CO2. In simpler terms, it removes a carboxyl group from CAIR to produce AIR and carbon dioxide, a key step in building purine nucleotides from scratch.
Why Is phosphoribosylaminoimidazole carboxylase activity Important in Cell Biology?
Phosphoribosylaminoimidazole carboxylase activity is essential for de novo purine biosynthesis, a pathway that supplies the nucleotides required for DNA replication, RNA transcription, and energy metabolism. Because rapidly dividing cancer cells depend heavily on purine synthesis, this activity has emerged as a promising target for anticancer therapy. Moreover, inherited defects in purine metabolism can cause severe disorders, and the enzyme's bifunctional nature in humans makes it a unique model for studying substrate channeling and metabolic channeling. Understanding GO:0004638 therefore bridges fundamental biochemistry, cancer biology, and therapeutic development.
• Provides a critical step in de novo purine biosynthesis, supplying precursors for DNA and RNA.
• PAICS, the human enzyme carrying this activity, is overexpressed in multiple cancers and correlates with poor prognosis.
• Knockdown of PAICS inhibits proliferation of breast cancer and gastric cancer cells.
• The carboxylase domain is a potential drug target because its inhibition selectively affects purine-dependent cancer cells.
• Loss of activity causes purine auxotrophy, as shown in CHO Ade-D mutants, linking it to the Ade-D locus.
• Substrate channeling between carboxylase and synthetase domains enhances metabolic efficiency and is a model for enzyme complex organization.
• The enzyme is a class II PurE, distinct from class I PurE found in bacteria, offering opportunities for selective antimicrobial targeting.
• Its activity can be monitored by 13C-bicarbonate incorporation, enabling flux analysis in cells and tissues.
• PAICS interacts with histone deacetylases and participates in DNA damage response, expanding its role beyond metabolism.
• CRISPR screens have identified PAICS as a therapeutic target for EGFR wild-type non-small cell lung cancer.
What Happens During phosphoribosylaminoimidazole carboxylase activity?
Substrate binding and decarboxylation
In simple terms: The enzyme grabs a molecule called CAIR and removes a carboxyl group from it.
The reaction begins with the binding of 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate (CAIR) to the active site of the carboxylase domain. In human PAICS, crystal structures have revealed the substrate-binding pocket and key residues that stabilize CAIR. The decarboxylation proceeds without the need for metal cofactors, characteristic of class II PurE enzymes. The product, 5-amino-1-(5-phospho-D-ribosyl)imidazole (AIR), is released along with carbon dioxide.
Product release and channeling
In simple terms: The product is handed directly to the next enzyme domain without floating away.
In the bifunctional human PAICS, the AIR product is channeled to the adjacent SAICAR synthetase domain, which immediately uses it in the next step of purine biosynthesis. Time-course analysis of 13C-bicarbonate incorporation provided evidence for this substrate channeling, showing that the intermediate does not equilibrate with the bulk solvent. This channeling enhances pathway efficiency and prevents loss of unstable intermediates.
Role in de novo purine biosynthesis
In simple terms: This step is one of many that build purine rings from scratch.
The carboxylase reaction is the sixth step in the ten-step de novo purine pathway. It follows the formation of CAIR and precedes the SAICAR synthetase step, which adds aspartate to AIR to form SAICAR. The pathway ultimately produces inosine monophosphate (IMP), the precursor of AMP and GMP. Thus, phosphoribosylaminoimidazole carboxylase activity is indispensable for purine nucleotide production.
Enzyme structure and oligomerization
In simple terms: The enzyme assembles into a larger complex of eight subunits to work properly.
Human PAICS forms a homooctamer, with each monomer containing an N-terminal carboxylase domain and a C-terminal synthetase domain. The octameric assembly is required for full catalytic activity and for substrate channeling. Crystal structures of human PAICS have provided detailed views of the active sites and the interface between domains.
Key Genes Involved in GO:0004638 phosphoribosylaminoimidazole carboxylase activity
The following genes and proteins are directly or indirectly involved in phosphoribosylaminoimidazole carboxylase activity and its broader purine biosynthesis pathway.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PAICS | Bifunctional enzyme with phosphoribosylaminoimidazole carboxylase and SAICAR synthetase activities | Overexpressed in cancers; target for knockout and inhibitor studies |
| ADE2 | Yeast homolog of the carboxylase domain; involved in purine biosynthesis | Model for studying purine pathway regulation and gene expression |
| GART | Trifunctional enzyme in de novo purine biosynthesis (GAR synthetase, AIR synthetase, GAR transformylase) | Upstream of PAICS; potential combination target |
| ATIC | Bifunctional enzyme (AICAR transformylase/IMP cyclohydrolase) in purine biosynthesis | Downstream of PAICS; links to purine nucleotide formation |
| PPAT | Phosphoribosyl pyrophosphate amidotransferase, first step of purine biosynthesis | Regulates flux into the pathway |
| PFAS | Phosphoribosylformylglycinamidine synthase, third step of purine biosynthesis | Potential metabolic context |
| ADSL | Adenylosuccinate lyase, involved in purine nucleotide interconversion | Related to purine metabolism disorders |
| HPRT1 | Hypoxanthine phosphoribosyltransferase 1, salvage pathway enzyme | Defects cause Lesch-Nyhan syndrome; contrasts with de novo pathway |
| PRPS1 | Phosphoribosyl pyrophosphate synthetase 1, supplies PRPP for purine synthesis | Mutations cause PRPS1 superactivity and gout |
| HDAC1 | Histone deacetylase 1, interacts with PAICS in DNA damage response | Links PAICS to epigenetic regulation |
| HDAC2 | Histone deacetylase 2, interacts with PAICS | Potential therapeutic target in gastric cancer |
| MYC | Oncogenic transcription factor that drives purine biosynthesis genes | Upstream regulator of PAICS expression |
| EGFR | Epidermal growth factor receptor; wild-type EGFR NSCLC depends on PAICS | Context for PAICS-targeted therapy |
| MTOR | mTOR kinase, regulates cell growth and metabolism | May influence purine pathway activity |
| PKM | Pyruvate kinase M, links glycolysis to biosynthetic pathways | Coordination of biosynthetic activity in myocardium |
| PFKM | Phosphofructokinase, muscle type; regulates glycolysis and biosynthetic coordination | Deep network tracing in myocardium |
| ATIC | AICAR transformylase/IMP cyclohydrolase | Downstream purine biosynthesis |
| GART | GAR synthetase/AIR synthetase/GAR transformylase | Upstream purine biosynthesis |
How Is phosphoribosylaminoimidazole carboxylase activity Regulated?
Phosphoribosylaminoimidazole carboxylase activity is regulated at multiple levels. Transcriptionally, PAICS expression is driven by oncogenic transcription factors such as MYC, which promotes purine biosynthesis to support proliferation. In cancer cells, PAICS is overexpressed, and its activity can be modulated by metabolic demand. The enzyme's bifunctional nature and substrate channeling suggest that its activity is intrinsically coordinated with the downstream SAICAR synthetase step. Additionally, PAICS interacts with histone deacetylases HDAC1 and HDAC2, linking its function to DNA damage response and epigenetic regulation. In the heart, phosphofructokinase (PFKM) coordinates biosynthetic pathway activity, including purine synthesis, as revealed by deep network tracing. These regulatory mechanisms ensure that purine production matches cellular needs for nucleotides and energy.
phosphoribosylaminoimidazole carboxylase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PAICS | Non-small cell lung cancer (EGFR wild-type) | CRISPR knockout in NSCLC cell lines; xenograft models |
| PAICS | Gastric cancer | Knockdown and overexpression in gastric cancer cells; HDAC interaction studies |
| PAICS | Breast cancer | Knockdown in breast cancer cell lines; proliferation assays |
| ADE2 | Purine auxotrophy (yeast model) | Yeast deletion mutants; complementation with human PAICS |
| PAICS | Myocardial biosynthetic coordination | In vivo deep network tracing in animal models |
PAICS in cancer
PAICS, the human enzyme with phosphoribosylaminoimidazole carboxylase activity, is overexpressed in several cancers. In EGFR wild-type non-small cell lung cancer, a genome-scale CRISPR-Cas9 screen identified PAICS as a therapeutic target, and its knockout suppressed tumor growth. In gastric cancer, PAICS contributes to carcinogenesis and participates in DNA damage response by interacting with HDAC1/2. Knockdown of PAICS in breast cancer cell lines inhibited malignant proliferation. These findings highlight PAICS as a promising anticancer target.
Purine auxotrophy and metabolic disorders
Loss of phosphoribosylaminoimidazole carboxylase activity leads to purine auxotrophy, as demonstrated in Chinese hamster ovary Ade-D mutants, which require exogenous purines for growth. The genetic locus correcting this defect maps to human chromosome 4, where PAICS is located. Inborn errors in purine metabolism can cause severe neurological and immunological disorders, although specific PAICS mutations in human disease are not well characterized.
PAICS in myocardial metabolism
In the myocardium, phosphofructokinase-mediated coordination of biosynthetic pathway activity, including purine synthesis, has been revealed by in vivo deep network tracing. This suggests that phosphoribosylaminoimidazole carboxylase activity may be part of a larger metabolic network that adjusts to cardiac energy demands.
From phosphoribosylaminoimidazole carboxylase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does PAICS loss inhibit tumor growth? | CRISPR knockout in cancer cell lines and mouse xenografts |
| What is the effect of a catalytic-dead PAICS mutant? | Point mutation (e.g., active-site residues) knock-in via CRISPR |
| How does PAICS substrate channeling work? | Tagged knock-in of PAICS domains for structural and flux studies |
| Does PAICS overexpression drive proliferation? | CRISPR-mediated overexpression in normal or cancer cells |
| What genes interact with PAICS in DNA damage response? | Knockout followed by proteomics and interaction studies |
| How does PAICS activity affect purine flux in vivo? | Metabolic tracing with 13C-bicarbonate in animal models |
How to Study the phosphoribosylaminoimidazole carboxylase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Coupled enzymatic assay | Carboxylase activity by NADH or NADPH consumption | Purification and kinetic characterization |
| X-ray crystallography | Three-dimensional structure of PAICS with substrates | Mechanistic studies and inhibitor design |
| 13C-bicarbonate incorporation | Flux through de novo purine synthesis | Substrate channeling and metabolic flux |
| CRISPR-Cas9 knockout | Loss-of-function effects on proliferation | Target validation in cancer cells |
| RNA-seq | Transcriptional changes after PAICS perturbation | Pathway analysis and off-target effects |
| Proteomics/co-immunoprecipitation | Protein-protein interactions (e.g., HDAC1/2) | DNA damage response studies |
| Deep network tracing | In vivo metabolic coordination | Myocardial biosynthetic pathway activity |
| Proliferation assays | Cell growth and viability | Cancer cell line studies |
Enzymatic assays for carboxylase activity
Phosphoribosylaminoimidazole carboxylase activity can be measured using coupled enzymatic assays that monitor the conversion of CAIR to AIR, often by spectrophotometric or radiometric methods. Purification of the bifunctional complex from natural sources has been described, enabling direct biochemical characterization.
Structural biology and substrate binding
Crystal structures of human PAICS have revealed substrate and product binding, providing insights into the catalytic mechanism and guiding inhibitor design. These studies are complemented by time-course analysis of 13C-bicarbonate incorporation to detect substrate channeling.
CRISPR screening and functional genomics
Genome-scale CRISPR-Cas9 screens have identified PAICS as a therapeutic target in EGFR wild-type non-small cell lung cancer. Such screens can be combined with RNA-seq and proteomics to dissect downstream effects.
Metabolic flux analysis
13C-bicarbonate incorporation followed by mass spectrometry allows measurement of de novo purine synthesis flux, revealing the contribution of phosphoribosylaminoimidazole carboxylase activity in cells and tissues. In vivo deep network tracing has been used to study biosynthetic coordination in the myocardium.
How CRISPR Can Be Used to Study GO:0004638 phosphoribosylaminoimidazole carboxylase activity
Knockout
CRISPR-Cas9 knockout of PAICS has been used to demonstrate its essential role in cancer cell proliferation. In EGFR wild-type non-small cell lung cancer, PAICS knockout suppressed tumor growth, validating it as a therapeutic target. Similarly, knockdown of PAICS in breast and gastric cancer cells inhibited malignant proliferation. Knockout models are valuable for assessing purine auxotrophy and metabolic dependencies.
Point Mutation
Point mutations in the carboxylase active site of PAICS can be introduced via CRISPR to dissect catalytic residues and separate carboxylase from synthetase activities. Such models help determine whether the enzymatic activity is required for cancer cell growth or whether non-catalytic functions dominate.
Knock-in
Tagged knock-in of PAICS (e.g., with FLAG or GFP) enables affinity purification and interaction studies, as well as live-cell imaging of the bifunctional enzyme. Knock-in of substrate-channeling mutants can reveal the importance of domain communication.
Overexpression
CRISPR-mediated overexpression of PAICS can model the elevated levels seen in cancers and test whether increased carboxylase activity drives proliferation or DNA damage resistance. Overexpression in normal cells can also reveal metabolic rewiring.
How EDITGENE Supports phosphoribosylaminoimidazole carboxylase activity Research
Researchers studying phosphoribosylaminoimidazole carboxylase activity-related genes often need to determine whether a candidate gene is causally involved in purine metabolism, cancer proliferation, or drug response. Precise genetic models are essential to move from correlation to causation, and CRISPR-based editing provides the necessary toolkit.
Contact EDITGENE today to design your custom CRISPR model for phosphoribosylaminoimidazole carboxylase activity research.
Frequently Asked Questions About phosphoribosylaminoimidazole carboxylase activity
What is phosphoribosylaminoimidazole carboxylase activity?
It is an enzymatic activity (GO:0004638) that converts CAIR to AIR and CO2 in de novo purine biosynthesis, carried out by the PAICS enzyme in humans.
What genes are involved in phosphoribosylaminoimidazole carboxylase activity?
The primary gene is PAICS, which encodes a bifunctional enzyme with both carboxylase and SAICAR synthetase activities. Other pathway genes include GART, ATIC, and PPAT.
What is the role of PAICS in cancer?
PAICS is overexpressed in several cancers, including lung, gastric, and breast cancer, and its knockdown inhibits proliferation, making it a therapeutic target.
How is phosphoribosylaminoimidazole carboxylase activity measured?
It can be measured by coupled enzymatic assays, 13C-bicarbonate incorporation, or structural studies of PAICS.
What diseases are associated with PAICS mutations?
PAICS mutations are not well characterized in human disease, but loss of activity causes purine auxotrophy in cell models. PAICS overexpression is linked to cancer.
What is the difference between class I and class II PurE?
Class II PurE, found in humans, is metal-independent and part of a bifunctional enzyme, while class I PurE is found in bacteria and requires metal cofactors.
How does substrate channeling work in PAICS?
The AIR product of the carboxylase domain is directly transferred to the synthetase domain without equilibrating with the solvent, enhancing pathway efficiency.
Can CRISPR be used to study phosphoribosylaminoimidazole carboxylase activity?
Yes, CRISPR knockout, point mutation, and overexpression models are widely used to dissect PAICS function in cancer and metabolism.
What is the clinical relevance of PAICS in lung cancer?
A genome-scale CRISPR screen identified PAICS as a therapeutic target for EGFR wild-type non-small cell lung cancer.
Where is PAICS located in the cell?
PAICS is a cytosolic enzyme, consistent with its role in de novo purine biosynthesis.
Conclusion
Phosphoribosylaminoimidazole carboxylase activity (GO:0004638) is a fundamental enzymatic step in de novo purine biosynthesis, catalyzed by the bifunctional enzyme PAICS in humans. Its role in cancer proliferation and metabolic reprogramming has made it an attractive therapeutic target, with CRISPR-based models providing critical causal insights. Continued research into its structure, regulation, and interactions will advance both basic metabolism and anticancer drug development.
References
- 1. Barton JW et al.. 1991. Mapping of a locus correcting lack of phosphoribosylaminoimidazole carboxylase activity in Chinese hamster ovary cell Ade-D mutants to human chromosome 4.. Genomics 9(2):314-21 PMID: 2004782
- 2. Li Y et al.. 2024. Genome-scale CRISPR-Cas9 screen identifies PAICS as a therapeutic target for EGFR wild-type non-small cell lung cancer.. MedComm (2020) 5(3):e483 PMID: 38463398
- 3. Humble RW et al.. 1991. Rapid purification of a bifunctional protein complex possessing phosphoribosylaminoimidazole carboxylase (EC 4.1.1.21) and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (EC 6.3.2.6) activities.. Adv Exp Med Biol 309B:223-7 PMID: 1781372
- 4. Fulghum KL et al.. 2022. In vivo deep network tracing reveals phosphofructokinase-mediated coordination of biosynthetic pathway activity in the myocardium.. J Mol Cell Cardiol 162:32-42 PMID: 34487754
- 5. Škerlová J et al.. 2020. Crystal structures of human PAICS reveal substrate and product binding of an emerging cancer target.. J Biol Chem 295(33):11656-11668 PMID: 32571877
- 6. Huang N et al.. 2020. PAICS contributes to gastric carcinogenesis and participates in DNA damage response by interacting with histone deacetylase 1/2.. Cell Death Dis 11(7):507 PMID: 32632107
- 7. Meng M et al.. 2018. Knockdown of PAICS inhibits malignant proliferation of human breast cancer cell lines.. Biol Res 51(1):24 PMID: 30097015
- 8. Shon H et al.. 2022. Evidence Supporting Substrate Channeling between Domains of Human PAICS: A Time-Course Analysis of (13)C-Bicarbonate Incorporation.. Biochemistry 61(7):575-582 PMID: 35285625
