Pfkfb4 encodes the testis isozyme of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), a homodimeric bifunctional enzyme with an N-terminal kinase domain and a C-terminal phosphatase domain of the phosphoglycerate mutase fold. The kinase domain phosphorylates fructose 6-phosphate with ATP to form fructose 2,6-bisphosphate, and the phosphatase domain hydrolyses fructose 2,6-bisphosphate back to fructose 6-phosphate; the recombinant rat enzyme has about fourfold more kinase than phosphatase activity. Fructose 2,6-bisphosphate is a potent allosteric activator of phosphofructokinase-1 and inhibitor of fructose-1,6-bisphosphatase, so the enzyme sets the balance between glycolytic and gluconeogenic flux. The cytosolic enzyme is expressed mainly in testis, and unlike the liver and heart isozymes it is phosphorylated by protein kinase C but not by cAMP-dependent protein kinase.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005829 cytosol | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: cytosol is retained as contextual support for Pfkfb4, but it is not the core function (IBA, GO_REF:0000033). Reason: cytosol records localization or complex context for Pfkfb4, not the defining molecular function. Supporting Evidence: UniProtKB:P25114 FUNCTION: Synthesis and degradation of fructose 2,6-bisphosphate. file:rat/Pfkfb4/Pfkfb4-deep-research-falcon.md Across eukaryotes including mammals, PFK-2/FBPase-2 activities are described as **cytosolic**. |
| GO:0003873 6-phosphofructo-2-kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Pfkfb4's bifunctional synthesis and degradation of fructose 2,6-bisphosphate supports retaining 6-phosphofructo-2-kinase activity as a direct annotation (IBA, GO_REF:0000033). Reason: 6-phosphofructo-2-kinase activity directly matches Pfkfb4's documented role in bifunctional synthesis and degradation of fructose 2,6-bisphosphate. Supporting Evidence: UniProtKB:P25114 FUNCTION: Synthesis and degradation of fructose 2,6-bisphosphate. file:rat/Pfkfb4/Pfkfb4-deep-research-falcon.md Fructose-2,6-bisphosphate is produced from fructose-6-phosphate by **6-phosphofructo-2-kinase (PFK-2; EC 2.7.1.105)** and hydrolyzed by **fructose-2,6-bisphosphatase (FBPase-2; EC 3.1.3.46)**; in mammals these opposing activities occur within the same bifunctional PFKFB enzymes. |
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: catalytic activity captures part of Pfkfb4 biology, but a more specific replacement better represents the direct role (IEA, GO_REF:0000002). Reason: catalytic activity is directionally related to Pfkfb4 but should be replaced by the more specific term(s): 6-phosphofructo-2-kinase activity, fructose-2,6-bisphosphate 2-phosphatase activity. Proposed replacements: 6-phosphofructo-2-kinase activity fructose-2,6-bisphosphate 2-phosphatase activity Supporting Evidence: UniProtKB:P25114 FUNCTION: Synthesis and degradation of fructose 2,6-bisphosphate. |
| GO:0003873 6-phosphofructo-2-kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Pfkfb4's bifunctional synthesis and degradation of fructose 2,6-bisphosphate supports retaining 6-phosphofructo-2-kinase activity as a direct annotation (IEA, GO_REF:0000120). Reason: 6-phosphofructo-2-kinase activity directly matches Pfkfb4's documented role in bifunctional synthesis and degradation of fructose 2,6-bisphosphate. Supporting Evidence: UniProtKB:P25114 FUNCTION: Synthesis and degradation of fructose 2,6-bisphosphate. |
| GO:0004331 fructose-2,6-bisphosphate 2-phosphatase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Pfkfb4's bifunctional synthesis and degradation of fructose 2,6-bisphosphate supports retaining fructose-2,6-bisphosphate 2-phosphatase activity as a direct annotation (IEA, GO_REF:0000120). Reason: fructose-2,6-bisphosphate 2-phosphatase activity directly matches Pfkfb4's documented role in bifunctional synthesis and degradation of fructose 2,6-bisphosphate. Supporting Evidence: UniProtKB:P25114 FUNCTION: Synthesis and degradation of fructose 2,6-bisphosphate. file:rat/Pfkfb4/Pfkfb4-deep-research-falcon.md Fructose-2,6-bisphosphate is produced from fructose-6-phosphate by **6-phosphofructo-2-kinase (PFK-2; EC 2.7.1.105)** and hydrolyzed by **fructose-2,6-bisphosphatase (FBPase-2; EC 3.1.3.46)**; in mammals these opposing activities occur within the same bifunctional PFKFB enzymes. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: ATP binding is retained as contextual support for Pfkfb4, but it is not the core function (IEA, GO_REF:0000002). Reason: ATP binding records cofactor, substrate, or interaction context for Pfkfb4, but the curated core role is bifunctional synthesis and degradation of fructose 2,6-bisphosphate. ATP is the phosphate donor of the kinase reaction. Supporting Evidence: UniProtKB:P25114 KM=270 uM for ATP |
| GO:0006000 fructose metabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: Pfkfb4's bifunctional synthesis and degradation of fructose 2,6-bisphosphate supports retaining fructose metabolic process as a direct annotation (IEA, GO_REF:0000002). Reason: fructose metabolic process is a direct process-level consequence of Pfkfb4's documented role in bifunctional synthesis and degradation of fructose 2,6-bisphosphate. GOA no longer carries this row (2026-10 refresh); the more specific fructose 2,6-bisphosphate metabolic process rows remain. Supporting Evidence: UniProtKB:P25114 FUNCTION: Synthesis and degradation of fructose 2,6-bisphosphate. |
| GO:0006003 fructose 2,6-bisphosphate metabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: Pfkfb4's bifunctional synthesis and degradation of fructose 2,6-bisphosphate supports retaining fructose 2,6-bisphosphate metabolic process as a direct annotation (IEA, GO_REF:0000002). Reason: fructose 2,6-bisphosphate metabolic process is a direct process-level consequence of Pfkfb4's documented role in bifunctional synthesis and degradation of fructose 2,6-bisphosphate. Supporting Evidence: UniProtKB:P25114 FUNCTION: Synthesis and degradation of fructose 2,6-bisphosphate. |
| GO:0003873 6-phosphofructo-2-kinase activity | ISO GO_REF:0000121 | ACCEPT | Summary: Pfkfb4's bifunctional synthesis and degradation of fructose 2,6-bisphosphate supports retaining 6-phosphofructo-2-kinase activity as a direct annotation (ISO, GO_REF:0000121). Reason: 6-phosphofructo-2-kinase activity directly matches Pfkfb4's documented role in bifunctional synthesis and degradation of fructose 2,6-bisphosphate. The ISO donor is human PFKFB4 (Q16877); the transfer is consistent with the rat IDA row from PMID:1651918. Supporting Evidence: UniProtKB:P25114 FUNCTION: Synthesis and degradation of fructose 2,6-bisphosphate. |
| GO:0006003 fructose 2,6-bisphosphate metabolic process | ISS GO_REF:0000114 | ACCEPT | Summary: Pfkfb4's bifunctional synthesis and degradation of fructose 2,6-bisphosphate supports retaining fructose 2,6-bisphosphate metabolic process as a direct annotation (ISS, GO_REF:0000114). Reason: fructose 2,6-bisphosphate metabolic process is a direct process-level consequence of Pfkfb4's documented role in bifunctional synthesis and degradation of fructose 2,6-bisphosphate. Supporting Evidence: UniProtKB:P25114 FUNCTION: Synthesis and degradation of fructose 2,6-bisphosphate. |
| GO:0006094 gluconeogenesis | NAS PMID:15581487 Regulation of the regulatory enzyme, 6-phosphofructo-2-kinas... | KEEP AS NON CORE | Summary: gluconeogenesis is retained as contextual support for Pfkfb4, but it is not the core function (NAS, PMID:15581487). Reason: gluconeogenesis is biologically related to Pfkfb4, but it is broader, downstream, or regulatory context relative to bifunctional synthesis and degradation of fructose 2,6-bisphosphate. Supporting Evidence: PMID:15581487 Regulation of the regulatory enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. file:rat/Pfkfb4/Pfkfb4-deep-research-falcon.md At **submicromolar concentrations**, it stimulates glycolysis and inhibits gluconeogenesis by: |
| GO:0006096 glycolytic process | NAS PMID:15581487 Regulation of the regulatory enzyme, 6-phosphofructo-2-kinas... | KEEP AS NON CORE | Summary: glycolytic process is retained as contextual support for Pfkfb4, but it is not the core function (NAS, PMID:15581487). Reason: glycolytic process is biologically related to Pfkfb4, but it is broader, downstream, or regulatory context relative to bifunctional synthesis and degradation of fructose 2,6-bisphosphate. Supporting Evidence: PMID:15581487 Regulation of the regulatory enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. file:rat/Pfkfb4/Pfkfb4-deep-research-falcon.md At the pathway level, PFKFB4 regulates **glycolytic flux** by controlling the concentration of **F-2,6-BP**, which directly tunes PFK1 activity. |
| GO:0043540 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase complex | IPI PMID:8805587 The crystal structure of the bifunctional enzyme 6-phosphofr... | KEEP AS NON CORE | Summary: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase complex is retained as contextual support for Pfkfb4, but it is not the core function (IPI, PMID:8805587). Reason: 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase complex records localization or complex context for Pfkfb4, not the defining molecular function. Supporting Evidence: PMID:8805587 RESULTS: We have determined the 2.0 A crystal structure of the rat testis isozyme of this bifunctional enzyme. file:rat/Pfkfb4/Pfkfb4-deep-research-falcon.md PFKFB enzymes are **bifunctional homodimers** with **two independent catalytic domains**: an **N-terminal kinase (PFK-2)** domain that synthesizes F-2,6-BP and a **C-terminal phosphatase (FBPase-2)** domain that degrades F-2,6-BP. |
| GO:0003873 6-phosphofructo-2-kinase activity | IDA PMID:1651918 Molecular cloning of the DNA and expression and characteriza... | ACCEPT | Summary: Pfkfb4's bifunctional synthesis and degradation of fructose 2,6-bisphosphate supports retaining 6-phosphofructo-2-kinase activity as a direct annotation (IDA, PMID:1651918). Reason: 6-phosphofructo-2-kinase activity directly matches Pfkfb4's documented role in bifunctional synthesis and degradation of fructose 2,6-bisphosphate. Supporting Evidence: PMID:1651918 We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. PMID:1651918 The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. |
| GO:0004331 fructose-2,6-bisphosphate 2-phosphatase activity | IDA PMID:1651918 Molecular cloning of the DNA and expression and characteriza... | ACCEPT | Summary: Pfkfb4's bifunctional synthesis and degradation of fructose 2,6-bisphosphate supports retaining fructose-2,6-bisphosphate 2-phosphatase activity as a direct annotation (IDA, PMID:1651918). Reason: fructose-2,6-bisphosphate 2-phosphatase activity directly matches Pfkfb4's documented role in bifunctional synthesis and degradation of fructose 2,6-bisphosphate. Supporting Evidence: PMID:1651918 We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. PMID:1651918 The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. |
| GO:0004331 fructose-2,6-bisphosphate 2-phosphatase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Fructose-2,6-bisphosphate 2-phosphatase activity is inherited from the PFKFB PAINT node PTN000064403, whose descendants include the mammalian PFKFB isozymes and yeast and plant bifunctional enzymes. Rat Pfkfb4 is inside that clade and its own phosphatase activity has been measured directly (PMID:1651918), so the IBA agrees with the experimental evidence. Reason: The C-terminal FBPase-2 domain is one of the two defining catalytic activities of PFKFB4. The recombinant rat enzyme hydrolyses fructose 2,6-bisphosphate (Km 21 uM), at lower specific activity than its kinase. No target-specific loss of the phosphatase is known. Supporting Evidence: UniProtKB:P25114 Reaction=beta-D-fructose 2,6-bisphosphate + H2O = beta-D-fructose 6- phosphate + phosphate PMID:1651918 The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. |
| GO:0006003 fructose 2,6-bisphosphate metabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: Fructose 2,6-bisphosphate metabolic process is inherited from the PFKFB PAINT node PTN000743282. Pfkfb4 both synthesizes and degrades fructose 2,6-bisphosphate, so it directly carries out this process. Reason: The process term is the direct process-level reading of the two catalytic activities measured for the rat enzyme (PMID:1651918), and matches the accepted IEA and ISS rows for the same term. Supporting Evidence: UniProtKB:P25114 FUNCTION: Synthesis and degradation of fructose 2,6-bisphosphate. PMID:1651918 The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. |
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