ENO3 encodes beta-enolase (muscle-specific enolase, MSE), the beta subunit of the glycolytic metalloenzyme enolase. It catalyses the penultimate step of glycolysis: the Mg2+-dependent, reversible dehydration of 2-phospho-D-glycerate (2-PG) to phosphoenolpyruvate (PEP) plus water (EC 4.2.1.11); the reverse hydration reaction operates in gluconeogenesis. Mammalian enolase functions as a dimer assembled from three tissue-specific subunits - alpha (ENO1, ubiquitous), beta (ENO3, muscle) and gamma (ENO2, neuronal) - which form homodimers and heterodimers; in striated muscle ENO3 forms alpha/beta heterodimers and beta/beta homodimers. The enzyme acts in the cytosol, with an additional sarcomeric pool localized to the Z line and, by similarity, the M band. Loss-of-function ENO3 variants cause glycogen storage disease type XIII (GSD13, beta-enolase deficiency), a metabolic myopathy of distal glycolysis characterized by exercise-induced myalgia, muscle weakness and fatigability, raised serum creatine kinase, and focal sarcoplasmic glycogen accumulation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004634 phosphopyruvate hydratase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetically-inferred (IBA) assignment of phosphopyruvate hydratase (enolase, EC 4.2.1.11) activity, the defining molecular function of the enolase family and the core catalytic function of ENO3. Beta-enolase converts 2-phosphoglycerate to phosphoenolpyruvate in glycolysis and the reverse in gluconeogenesis. Reason: This is the core molecular function of ENO3, at the correct level of specificity, supported by UniProt, EC/RHEA mapping, ISS from mouse ortholog, and multiple TAS Reactome annotations. The IBA is well supported across the enolase orthology group. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt Enolase that catalyzes the conversion of 2-phosphoglycerate file:human/ENO3/ENO3-uniprot.txt Reaction=(2R)-2-phosphoglycerate = phosphoenolpyruvate + H2O |
| GO:0006096 glycolytic process | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetically-inferred involvement in glycolysis. Enolase catalyses the ninth step of glycolysis (2-PG to PEP), a core biological process for ENO3. Reason: Correct and core. ENO3 is a canonical glycolytic enzyme; the muscle beta-enolase supplies the high glycolytic flux of striated muscle. Supported by UniProt PATHWAY. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt glyceraldehyde 3-phosphate: step 4/5 |
| GO:0000015 phosphopyruvate hydratase complex | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetically-inferred assignment to the phosphopyruvate hydratase (enolase) complex - the catalytically active enolase dimer. ENO3 acts as a beta/beta homodimer or an alpha/beta heterodimer with ENO1. Reason: Enolase is obligately dimeric and Mg2+ stabilizes the dimer; the complex term accurately captures ENO3's quaternary organization. This is where the informative content of the bare protein-binding IPIs (ENO1-ENO3) is properly represented. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt SUBUNIT: Mammalian enolase is composed of 3 isozyme subunits, alpha, |
| GO:0000015 phosphopyruvate hydratase complex | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO (IPR000941 Enolase) electronic assignment to the enolase dimer complex. Duplicates the IBA complex annotation. Reason: Correct InterPro-based mapping; consistent with the IBA part_of annotation and with the obligately dimeric nature of enolase. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt SUBUNIT: Mammalian enolase is composed of 3 isozyme subunits, alpha, |
| GO:0000287 magnesium ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO (IPR000941 Enolase) electronic assignment of magnesium ion binding. Mg2+ is the essential catalytic cofactor of enolase; the structure of P13929 has annotated Mg2+-binding residues (Ser-245, Asp-293, Asp-318). Reason: Well supported. Mg2+ is required for catalysis and for stabilizing the enolase dimer, and UniProt lists explicit Mg2+ BINDING sites in ENO3. A genuine cofactor function; retained as core because catalysis is Mg2+-dependent. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt Note=Mg(2+) is required for catalysis and for stabilizing the dimer |
| GO:0004634 phosphopyruvate hydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of phosphopyruvate hydratase activity via combined IEA methods (EC 4.2.1.11, RHEA:10164, InterPro, mouse ortholog). Duplicates the core enolase molecular function. Reason: Correct EC/RHEA/InterPro mapping to the core catalytic function of ENO3. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt Reaction=(2R)-2-phosphoglycerate = phosphoenolpyruvate + H2O |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic assignment of cytoplasmic localization from the UniProt subcellular location vocabulary. Enolase is a soluble cytoplasmic/cytosolic enzyme. Reason: Correct but general; the more specific GO:0005829 cytosol (IDA, HPA) is the preferred core location. Kept as an accurate parent-level localization. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. Note=Localized to the Z line |
| GO:0006094 gluconeogenesis | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA electronic assignment of involvement in gluconeogenesis. Enolase catalyses the reversible 2-PG/PEP interconversion; running in the hydration (PEP to 2-PG) direction, it participates in the gluconeogenic pathway. Reason: Biologically correct - the enolase reaction is reversible and operates in both glycolysis and gluconeogenesis, as stated explicitly in the UniProt FUNCTION line. Note that muscle is not a major gluconeogenic tissue, so this is a shared enzymatic capability rather than the dominant physiological role of ENO3, but the annotation is not wrong. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt to phosphoenolpyruvate in glycolysis and the reverse reaction in |
| GO:0006096 glycolytic process | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment (InterPro + UniPathway UPA00109) of involvement in glycolysis. Duplicates the IBA glycolytic process annotation. Reason: Correct core biological process; consistent with the IBA and Reactome annotations. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt glyceraldehyde 3-phosphate: step 4/5 |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | MARK AS OVER ANNOTATED | Summary: IntAct-curated physical interaction (BioPlex AP-MS interactome) of ENO3 with ENO1 (UniProtKB:P06733), i.e. the alpha/beta enolase heterodimer. Annotated with the uninformative generic term protein binding. Reason: Bare "protein binding" (GO:0005515) is uninformative and per curation guidelines should not be treated as a core function. The biologically meaningful content - the ENO1-ENO3 (alpha/beta) association - is properly represented by GO:0000015 phosphopyruvate hydratase complex. The interaction itself is a real, high-throughput AP-MS observation, so it is not removed, only flagged as over-annotation. Supporting Evidence: PMID:33961781 affinity purification of 10,128 human proteins-half the proteome-in 293T cells and includes 118,162 interactions among 14,586 proteins |
| GO:0005515 protein binding | IPI PMID:35271311 OpenCell: Endogenous tagging for the cartography of human ce... | MARK AS OVER ANNOTATED | Summary: IntAct-curated physical interaction (OpenCell endogenous-tagging IP-MS) of ENO3 with ENO1 (UniProtKB:P06733), again the alpha/beta enolase heterodimer, annotated with the generic term protein binding. Reason: As for the BioPlex IPI, bare "protein binding" is uninformative; the ENO1-ENO3 interaction is captured more specifically by GO:0000015 phosphopyruvate hydratase complex. The interaction is a genuine large-scale IP-MS observation, so it is flagged as over-annotation rather than removed. Supporting Evidence: PMID:35271311 systematically map the localization and interactions of human proteins |
| GO:0005829 cytosol | IEA GO_REF:0000107 | ACCEPT | Summary: Ensembl-Compara electronic transfer (from mouse ortholog) of active localization in the cytosol - the compartment where glycolysis, and thus the enolase reaction, occurs. Reason: Correct and core; the cytosol is where ENO3 performs its glycolytic function. The is_active_in qualifier is appropriate for a soluble metabolic enzyme. Corroborated by the HPA IDA cytosol annotation. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. Note=Localized to the Z line |
| GO:0061621 canonical glycolysis | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment (ARBA + mouse ortholog) of involvement in canonical glycolysis, the ATP-generating glucose-to-pyruvate route. This is a more specific child of glycolytic process describing exactly the pathway ENO3 participates in. Reason: Correct and appropriately specific; ENO3 catalyses the penultimate step of canonical glycolysis. Consistent with the Reactome TAS canonical glycolysis annotation. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt glyceraldehyde 3-phosphate: step 4/5 |
| GO:0006094 gluconeogenesis | TAS Reactome:R-HSA-70263 | ACCEPT | Summary: Reactome traceable-author-statement placing ENO3 (as part of the enolase dimers) in the Gluconeogenesis pathway, catalysing the hydration of PEP to 2-PG. Reason: Biologically correct; the reversible enolase reaction operates in gluconeogenesis. Consistent with the ARBA IEA gluconeogenesis annotation and with UniProt FUNCTION. Shared enzymatic capability rather than the dominant muscle role, but not incorrect. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt to phosphoenolpyruvate in glycolysis and the reverse reaction in |
| GO:0061621 canonical glycolysis | TAS Reactome:R-HSA-70171 | ACCEPT | Summary: Reactome traceable-author-statement placing ENO3 in the Glycolysis pathway (canonical glycolysis). Reason: Correct and core; duplicates the IEA canonical glycolysis annotation with expert Reactome pathway curation. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt glyceraldehyde 3-phosphate: step 4/5 |
| GO:0004634 phosphopyruvate hydratase activity | TAS Reactome:R-HSA-70494 | ACCEPT | Summary: Reactome traceable-author-statement for the enolase reaction ("Enolase dimers (ENO1,2,3) convert PEP to 2PG"), assigning phosphopyruvate hydratase activity to ENO3. Reason: Expert-curated statement of the core catalytic function of ENO3; duplicates the IBA/IEA/ISS enolase MF annotations. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt Reaction=(2R)-2-phosphoglycerate = phosphoenolpyruvate + H2O |
| GO:0004634 phosphopyruvate hydratase activity | TAS Reactome:R-HSA-71660 | ACCEPT | Summary: Reactome traceable-author-statement ("Enolase dimers dehydrates 2PG") for the phosphopyruvate hydratase activity of ENO3. Reason: Expert-curated statement of the core enolase catalytic function; duplicates the other enolase MF annotations. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt Reaction=(2R)-2-phosphoglycerate = phosphoenolpyruvate + H2O |
| GO:0005829 cytosol | IDA GO_REF:0000052 | ACCEPT | Summary: Direct assay (Human Protein Atlas immunofluorescence) localizing ENO3 to the cytosol, the primary site of the glycolytic enolase reaction. Reason: Experimental, specific, and core. This is the preferred cellular-component annotation for ENO3 and is consistent with UniProt (Cytoplasm) and the Ensembl is_active_in cytosol annotation. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. Note=Localized to the Z line |
| GO:0005886 plasma membrane | IDA GO_REF:0000052 | KEEP AS NON CORE | Summary: Direct assay (Human Protein Atlas immunofluorescence) reporting a plasma-membrane signal for ENO3. Cell-surface enolase pools have been reported for enolases generally, but this is not the core cytosolic catalytic localization. Reason: Enolase is a well-known moonlighting cell-surface protein in several isozymes, so a plasma-membrane pool is plausible and the experimental HPA signal is retained; however it does not represent the core glycolytic function of ENO3 and is marked non-core. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. Note=Localized to the Z line |
| GO:0004634 phosphopyruvate hydratase activity | ISS GO_REF:0000024 | ACCEPT | Summary: Sequence-similarity-based transfer (from the rat/mouse ortholog UniProtKB:P15429) of phosphopyruvate hydratase activity to ENO3. Duplicates the core enolase MF. Reason: Correct ortholog-based transfer of the core catalytic function; the human ENO3 EC 4.2.1.11 assignment itself is ISS-inferred from P15429 in UniProt. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt Reaction=(2R)-2-phosphoglycerate = phosphoenolpyruvate + H2O |
| GO:0016020 membrane | IDA PMID:19433310 Cathepsin X cleaves the C-terminal dipeptide of alpha- and g... | MARK AS OVER ANNOTATED | Summary: CAFA-sourced direct-assay annotation to the generic term membrane, from a study of cathepsin X cleavage of enolase. The cited paper concerns alpha- and gamma-enolase neuronal biology, not muscle beta-enolase. Reason: "Membrane" (GO:0016020) is a very general cellular-component term and does not represent the core cytosolic localization of ENO3. The supporting paper studies alpha/gamma-enolase (neurotrophic, cell-surface enolase), so its relevance to muscle beta-enolase is peripheral. Flagged as over-annotation rather than removed, since a moonlighting cell-surface/membrane enolase pool is documented for the family. Supporting Evidence: PMID:19433310 we identify isozymes alpha- and gamma-enolases as targets for cathepsin X |
| GO:0070062 extracellular exosome | HDA PMID:23533145 In-depth proteomic analyses of exosomes isolated from expres... | MARK AS OVER ANNOTATED | Summary: High-throughput proteomic detection of ENO3 in exosomes isolated from expressed prostatic secretions in urine. A bulk secretome/exosome proteomics survey. Reason: Glycolytic enzymes, including enolases, are ubiquitously detected in exosome/secretome proteomes, and such detections rarely reflect a dedicated biological function. This is not a core localization for muscle beta-enolase and is flagged as over-annotation. Kept (not removed) as it is a valid mass-spectrometry observation. Supporting Evidence: PMID:23533145 exosome preparations were characterized by a shotgun proteomics procedure |
| GO:0005576 extracellular region | HDA PMID:22664934 Comparison of tear protein levels in breast cancer patients ... | MARK AS OVER ANNOTATED | Summary: High-throughput proteomic detection of ENO3 in tear fluid (a comparative breast-cancer tear-proteome study). A bulk body-fluid proteomics survey. Reason: Detection of an abundant cytosolic glycolytic enzyme in a body fluid does not establish a dedicated extracellular function; such findings are common contaminant/secretome observations. Not a core localization for ENO3, so flagged as over-annotation while retaining the experimental record. Supporting Evidence: PMID:22664934 semi-quantitative comparison of tear protein levels in cancer (CA) and control |
| GO:0005829 cytosol | TAS Reactome:R-HSA-70494 | ACCEPT | Summary: Reactome traceable-author-statement localizing the ENO3-containing enolase reaction to the cytosol. Reason: Correct and core; the enolase reaction occurs in the cytosol. Duplicates the HPA IDA cytosol annotation with expert pathway curation. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. Note=Localized to the Z line |
| GO:0005829 cytosol | TAS Reactome:R-HSA-71660 | ACCEPT | Summary: Reactome traceable-author-statement localizing the ENO3 enolase reaction to the cytosol. Reason: Correct and core; consistent with the other cytosol/cytoplasm annotations. Supporting Evidence: file:human/ENO3/ENO3-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. Note=Localized to the Z line |
| GO:0004634 phosphopyruvate hydratase activity | TAS PMID:8513787 Structural features of the human gene for muscle-specific en... | ACCEPT | Summary: Traceable-author-statement (PINC) assigning phosphopyruvate hydratase activity to ENO3. The cited paper characterizes the human muscle-specific (beta) enolase gene and identifies it as the beta isoform of the glycolytic enzyme enolase. Reason: Core catalytic function; the paper explicitly identifies ENO3 as the beta/muscle-specific isoform of the glycolytic enzyme enolase. Duplicates the other enolase MF annotations. Supporting Evidence: PMID:8513787 the human gene for the beta or muscle-specific isoform of the glycolytic enzyme enolase |
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