GO:0017168 5-oxoprolinase (ATP-hydrolyzing) activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0017168 defines the ATP-dependent hydrolysis of 5-oxo-L-proline (pyroglutamate) to L-glutamate, a reaction that links glutathione recycling to amino acid homeostasis.
• The enzyme is a member of the N-terminal nucleophile (Ntn) hydrolase family and uses a covalent thioester intermediate for catalysis.
• 5-Oxoprolinase activity is widely distributed across prokaryotes and eukaryotes, with a conserved two-subunit architecture in many bacteria.
• Kinetic parameters and tissue distribution have been characterized in mammals, showing highest activity in kidney and liver.
• Bovine 5-oxo-L-prolinase has been purified and its cDNA cloned, revealing expression in coronary artery.
• The enzyme also accepts L-2-oxothiazolidine-4-carboxylic acid as a substrate, linking it to prodrug activation and cysteine delivery.
Description
5-Oxoprolinase (ATP-hydrolyzing) activity, encoded by GO:0017168, catalyzes the conversion of 5-oxo-L-proline (also known as pyroglutamate) to L-glutamate in an ATP-dependent manner. This reaction is a critical step in the gamma-glutamyl cycle, which governs glutathione turnover and amino acid transport. The enzyme is conserved from bacteria to humans, and its dysfunction has been linked to metabolic disorders and oxidative stress. Researchers study this activity to understand glutathione homeostasis, drug metabolism, and the role of pyroglutamate as a signaling molecule. The reaction consumes ATP and water, producing ADP, phosphate, and two protons, making it an energy-dependent hydrolase. Because 5-oxoprolinase is involved in the detoxification of pyroglutamate and the recycling of cysteine, it is a target for both fundamental enzymology and translational research.
5-oxoprolinase (ATP-hydrolyzing) activity At A Glance
| GO ID | GO:0017168 |
|---|---|
| GO term | 5-oxoprolinase (ATP-hydrolyzing) activity |
| Ontology | molecular_function |
| Synonym | 5-OPase activity; 5-oxo-L-prolinase activity; pyroglutamase (ATP-hydrolyzing) activity; L-pyroglutamate hydrolase activity |
| Major function | ATP-dependent hydrolysis of 5-oxo-L-proline to L-glutamate |
| Reaction | 5-oxo-L-proline + ATP + 2 H2O = L-glutamate + ADP + 2 H+ + phosphate |
| Cofactors | ATP; Mg2+ (implied by ATP-dependent hydrolase mechanism) |
| Subcellular location | Cytoplasm (inferred from eukaryotic studies) |
| Enzyme family | N-terminal nucleophile (Ntn) hydrolase family |
What Is GO:0017168?
According to the Gene Ontology, GO:0017168 (5-oxoprolinase (ATP-hydrolyzing) activity) is defined as the catalysis of the reaction: 5-oxo-L-proline + ATP + 2 H2O = L-glutamate + ADP + 2 H+ + phosphate. In other words, it is an ATP-powered enzyme that breaks down 5-oxo-L-proline into glutamate, consuming water and releasing energy as ADP and phosphate.
Why Is 5-oxoprolinase (ATP-hydrolyzing) activity Important in Cell Biology?
5-Oxoprolinase activity is essential for maintaining cellular glutamate and glutathione levels, which are central to redox balance, detoxification, and neurotransmission. Its ability to hydrolyze pyroglutamate prevents the accumulation of this metabolite, which can be neurotoxic at high concentrations. The enzyme also activates prodrugs such as L-2-oxothiazolidine-4-carboxylic acid, making it relevant to pharmacology. In mammals, tissue-specific expression patterns suggest specialized roles in kidney and liver metabolism. Understanding this activity helps researchers dissect the gamma-glutamyl cycle and its contributions to disease.
• Maintains glutamate homeostasis by recycling pyroglutamate.
• Supports glutathione synthesis and redox balance.
• Prevents accumulation of neurotoxic pyroglutamate.
• Activates prodrugs like L-2-oxothiazolidine-4-carboxylic acid for cysteine delivery.
• Expressed in kidney and liver, indicating roles in amino acid transport and detoxification.
• Detected in coronary artery, suggesting vascular functions.
• Conserved in prokaryotes, offering model systems for mechanistic studies.
• Potential target for metabolic disorders linked to glutathione dysregulation.
Molecular Mechanism of 5-oxoprolinase (ATP-hydrolyzing) activity
Substrate Binding and ATP Hydrolysis
In simple terms: The enzyme grabs 5-oxo-L-proline and uses ATP to power its breakdown.
5-Oxoprolinase binds 5-oxo-L-proline and ATP in a sequential manner. ATP hydrolysis provides the energy required for the cleavage of the substrate, yielding ADP and phosphate. The reaction also consumes two water molecules and releases two protons. Kinetic studies in mammals have determined the Km and Vmax for 5-oxo-L-proline, showing high affinity in kidney and liver tissues.
Covalent Catalysis via Thioester Intermediate
In simple terms: The enzyme forms a temporary bond with the substrate to break it down.
The catalytic mechanism involves a covalent thioester intermediate formed between a conserved cysteine residue and the substrate. This is characteristic of the N-terminal nucleophile (Ntn) hydrolase family, where the N-terminal residue acts as a nucleophile. The intermediate is subsequently resolved by water, releasing glutamate.
Substrate Specificity and Alternative Substrates
In simple terms: The enzyme prefers 5-oxo-L-proline but can also act on similar molecules.
While 5-oxo-L-proline is the primary substrate, the enzyme also hydrolyzes L-2-oxothiazolidine-4-carboxylic acid, a cysteine prodrug. This broad specificity suggests a role in xenobiotic metabolism. The enzyme from Paecilomyces varioti has been purified and shown to be ATP-dependent, confirming conservation of mechanism across fungi.
Structural Organization and Subunit Composition
In simple terms: The enzyme is built from two parts that work together.
In many prokaryotes, 5-oxoprolinase is composed of two subunits encoded by separate genes, forming a heterodimeric complex. The bovine enzyme has been purified as a single polypeptide, and its cDNA was cloned, revealing a multidomain architecture. The active site is located at the interface of the subunits or domains, with the N-terminal nucleophile playing a key role.
Regulation by ATP and Metal Ions
In simple terms: ATP and magnesium help the enzyme work efficiently.
The enzyme requires ATP and likely Mg2+ for optimal activity, as is typical for ATP-dependent hydrolases. The ATP-binding site is conserved across species. No allosteric regulators have been definitively identified, but the enzyme's activity is tied to cellular energy status.
Key Genes Involved in GO:0017168 5-oxoprolinase (ATP-hydrolyzing) activity
The following genes and proteins are directly involved in or regulate 5-oxoprolinase (ATP-hydrolyzing) activity.
| Gene | Major Role | Research Relevance |
|---|---|---|
| OPLAH | Encodes 5-oxoprolinase in humans | Mutations linked to 5-oxoprolinuria and metabolic disorders |
| OPLA | Prokaryotic 5-oxoprolinase subunit A | Model for structural and mechanistic studies |
| OPLB | Prokaryotic 5-oxoprolinase subunit B | Required for ATP-dependent catalysis |
| GGT1 | Gamma-glutamyl transpeptidase | Upstream of 5-oxoprolinase in gamma-glutamyl cycle |
| GGCT | Gamma-glutamyl cyclotransferase | Produces 5-oxo-L-proline for 5-oxoprolinase |
| GCLC | Glutamate-cysteine ligase catalytic subunit | Glutathione synthesis, linked to glutamate supply |
| GCLM | Glutamate-cysteine ligase modifier subunit | Regulates glutathione synthesis |
| GSS | Glutathione synthetase | Downstream of glutamate in glutathione synthesis |
| G6PD | Glucose-6-phosphate dehydrogenase | Provides NADPH for glutathione recycling |
| SLC7A11 | Cystine/glutamate antiporter | Influences glutamate and cysteine availability |
| OTC | Ornithine transcarbamylase | Urea cycle, related to glutamate metabolism |
| GLS | Glutaminase | Produces glutamate from glutamine |
| GLUD1 | Glutamate dehydrogenase 1 | Interconverts glutamate and alpha-ketoglutarate |
| GAD1 | Glutamate decarboxylase 1 | Produces GABA from glutamate |
| GAD2 | Glutamate decarboxylase 2 | Produces GABA from glutamate |
| EAAT1 | Glutamate transporter | Regulates extracellular glutamate |
| EAAT2 | Glutamate transporter | Regulates extracellular glutamate |
How Is 5-oxoprolinase (ATP-hydrolyzing) activity Regulated?
The activity of 5-oxoprolinase is primarily regulated by substrate availability and cellular energy status, as it requires ATP. No specific allosteric regulators have been identified, but its expression may be influenced by oxidative stress and glutathione demand. In mammals, tissue-specific expression patterns suggest hormonal or metabolic regulation, though direct evidence is limited.
5-oxoprolinase (ATP-hydrolyzing) activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| OPLAH | 5-Oxoprolinuria | OPLAH knockout cell line (e.g., HEK293) |
| OPLAH | Acetaminophen-induced toxicity | Primary hepatocytes with OPLAH knockdown |
| OPLAH | Cancer redox balance | Cancer cell lines with OPLAH overexpression |
| OPLA | Bacterial metabolism | E. coli knockout mutants |
| OPLB | Bacterial metabolism | E. coli knockout mutants |
5-Oxoprolinuria and Metabolic Disorders
Deficiency of 5-oxoprolinase activity leads to 5-oxoprolinuria, characterized by elevated urinary 5-oxo-L-proline and metabolic acidosis. This condition can result from inherited mutations in OPLAH or from acquired causes such as acetaminophen toxicity. Patients may present with neurological symptoms due to pyroglutamate accumulation.
Cancer and Oxidative Stress
Altered glutathione metabolism is a hallmark of many cancers, and 5-oxoprolinase supports glutathione recycling. High expression of OPLAH has been observed in some tumors, suggesting a role in maintaining redox balance. Targeting this enzyme could sensitize cancer cells to oxidative stress.
Neurodegeneration and Excitotoxicity
Pyroglutamate is a neuroactive metabolite, and its accumulation due to 5-oxoprolinase dysfunction may contribute to excitotoxicity. Glutamate, the product of the reaction, is a major excitatory neurotransmitter, linking this enzyme to neurological disorders.
From 5-oxoprolinase (ATP-hydrolyzing) activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does OPLAH loss cause pyroglutamate accumulation? | OPLAH knockout HEK293 cells |
| Does a point mutation in the catalytic cysteine abolish activity? | Point-mutant OPLAH knock-in cells |
| Can tagged OPLAH be used for localization studies? | Tagged knock-in (e.g., GFP-OPLAH) |
| Does OPLAH overexpression protect against oxidative stress? | OPLAH overexpression in cancer cell lines |
| Is OPLAH required for glutathione homeostasis? | OPLAH knockout in primary hepatocytes |
| Can CRISPR library screening identify synthetic lethal partners? | Genome-wide CRISPR knockout library in OPLAH-mutant cells |
How to Study the 5-oxoprolinase (ATP-hydrolyzing) activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Fluorimetric assay | Enzymatic activity | Tissue distribution studies |
| LC-MS metabolomics | 5-oxo-L-proline and glutamate levels | Metabolic profiling |
| Western blot | Protein expression | Tissue-specific expression |
| RT-PCR | mRNA levels | Tissue distribution |
| CRISPR knockout | Gene function | Loss-of-function studies |
| CRISPR knock-in | Tagged protein localization | Imaging and proteomics |
| Enzyme kinetics | Km, Vmax, kcat | Mechanistic studies |
Enzymatic Activity Assays
Fluorimetric assays using coupled enzymes can measure 5-oxoprolinase activity in tissue lysates, as demonstrated for mammalian tissues. These assays typically monitor NADH or NADPH consumption linked to the reaction.
Purification and Characterization
Purification from native sources, such as bovine kidney or Paecilomyces varioti, followed by kinetic analysis, provides insights into substrate specificity and cofactor requirements. Recombinant expression in E. coli is also used for prokaryotic enzymes.
Molecular Biology and Cloning
cDNA cloning and mRNA detection by Northern blot or RT-PCR have been used to study tissue distribution, as shown for bovine 5-oxo-L-prolinase. CRISPR-based knockout and knock-in models enable functional studies.
Metabolomics and Flux Analysis
Mass spectrometry-based metabolomics can quantify 5-oxo-L-proline and glutamate levels in cells and tissues, revealing flux through the gamma-glutamyl cycle. Isotope tracing can track glutamate production from pyroglutamate.
How CRISPR Can Be Used to Study GO:0017168 5-oxoprolinase (ATP-hydrolyzing) activity
Knockout
CRISPR knockout of OPLAH or bacterial opla/oplb genes can abolish 5-oxoprolinase activity, leading to pyroglutamate accumulation and glutathione depletion. These models are useful for studying metabolic consequences and synthetic lethality.
Point Mutation
Introducing point mutations in the catalytic cysteine or ATP-binding residues can dissect the mechanism of covalent catalysis and ATP hydrolysis. Such mutants are valuable for structure-function studies.
Knock-in
Knock-in of tagged OPLAH (e.g., GFP or FLAG) allows visualization and immunoprecipitation of the enzyme in live cells. This approach can reveal subcellular localization and interaction partners.
Overexpression
Overexpression of OPLAH in cell lines can enhance glutathione recycling and protect against oxidative stress. It is also used to study prodrug activation, such as L-2-oxothiazolidine-4-carboxylic acid.
How EDITGENE Supports 5-oxoprolinase (ATP-hydrolyzing) activity Research
Researchers studying 5-oxoprolinase (ATP-hydrolyzing) activity-related genes often need to determine whether a candidate gene is causally involved in metabolic pathways, disease phenotypes, or drug responses. EDITGENE provides a comprehensive suite of CRISPR-based services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for 5-oxoprolinase (ATP-hydrolyzing) activity research.
Frequently Asked Questions About 5-oxoprolinase (ATP-hydrolyzing) activity
What is 5-oxoprolinase (ATP-hydrolyzing) activity?
It is an enzymatic activity defined by GO:0017168 that catalyzes the ATP-dependent conversion of 5-oxo-L-proline to L-glutamate, consuming water and releasing ADP and phosphate.
What genes are involved in 5-oxoprolinase (ATP-hydrolyzing) activity?
The main gene in humans is OPLAH, while bacteria use opla and oplb. Other genes in the gamma-glutamyl cycle include GGT1, GGCT, GCLC, GCLM, and GSS.
What is the reaction catalyzed by 5-oxoprolinase?
The reaction is: 5-oxo-L-proline + ATP + 2 H2O = L-glutamate + ADP + 2 H+ + phosphate.
Where is 5-oxoprolinase found in the body?
It is most active in kidney and liver, and has also been detected in coronary artery.
What diseases are associated with 5-oxoprolinase deficiency?
Deficiency causes 5-oxoprolinuria, a metabolic disorder with elevated urinary pyroglutamate, and may contribute to oxidative stress-related diseases.
How can I study 5-oxoprolinase activity in the lab?
Common methods include fluorimetric enzyme assays, LC-MS metabolomics, Western blot, and CRISPR knockout/knock-in models.
What is the mechanism of 5-oxoprolinase?
It uses a covalent thioester intermediate and ATP hydrolysis to cleave 5-oxo-L-proline, typical of Ntn hydrolases.
Can 5-oxoprolinase activate prodrugs?
Yes, it can hydrolyze L-2-oxothiazolidine-4-carboxylic acid to release cysteine, making it relevant for prodrug design.
Is 5-oxoprolinase conserved in bacteria?
Yes, a widespread prokaryotic 5-oxoprolinase was recently discovered, often composed of two subunits.
What CRISPR models are available for 5-oxoprolinase research?
EDITGENE offers knockout, point mutation, knock-in, tagged knock-in, and overexpression models for OPLAH and related genes.
Conclusion
5-Oxoprolinase (ATP-hydrolyzing) activity, encoded by GO:0017168, is a key enzymatic step in glutathione recycling and glutamate homeostasis. Its mechanism, involving ATP-dependent covalent catalysis, is conserved across species and has implications for metabolic disorders, cancer, and neurodegeneration. Continued research using CRISPR models and advanced metabolomics will further illuminate its roles and therapeutic potential.
References
- 1. Niehaus TD et al.. 2017. Discovery of a widespread prokaryotic 5-oxoprolinase that was hiding in plain sight.. J Biol Chem 292(39):16360-16367 PMID: 28830929
- 2. Weber P et al.. 1999. Kinetic parameters and tissue distribution of 5-oxo-L-prolinase determined by a fluorimetric assay.. J Biochem Biophys Methods 38(1):71-82 PMID: 10078874
- 3. Watanabe T et al.. 2004. Bovine 5-oxo-L-prolinase: simple assay method, purification, cDNA cloning, and detection of mRNA in the coronary artery.. Biol Pharm Bull 27(3):288-94 PMID: 14993790
- 4. Mochizuki K. 1999. Purification and characterization of 5-oxo-L-prolinase from Paecilomyces varioti F-1, an ATP-dependent hydrolase active with L-2-oxothiazolidine-4-carboxylic acid.. Arch Microbiol 172(3):182-5 PMID: 10460889
