Enolase

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phosphopyruvate hydratase
ExPASy
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KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
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NCBIproteins
Enolase, N-terminal domain
SCOP2
1els / SCOPe / SUPFAM
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
Enolase
Crystal structure of dimeric beta human enolase ENO3.[2]
Identifiers
SymbolEnolase
PfamPF00113
InterProIPR000941
PROSITEPDOC00148
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary
PDBPDB: 1e9i​ PDB: 1ebg​ PDB: 1ebh​ PDB: 1els​ PDB: 1iyx​ PDB: 1l8p​ PDB: 1nel​ PDB: 1oep​ PDB: 1one​ PDB: 1p43​

Phosphopyruvate hydratase, usually known as enolase, is a

phosphoenolpyruvate (PEP), the ninth and penultimate step of glycolysis. The chemical reaction
is:

2-phospho-D-glycerate phosphoenolpyruvate + H2O

Phosphopyruvate hydratase belongs to the family of lyases, specifically the hydro-lyases, which cleave carbon-oxygen bonds. The systematic name of this enzyme is 2-phospho-D-glycerate hydro-lyase (phosphoenolpyruvate-forming).

The reaction is reversible, depending on environmental concentrations of substrates.

haemolytic anemia, while ENO3 deficiency is linked to glycogen storage disease type XIII
.

Isozymes

In humans there are three subunits of enolase,

isoenzymes: αα, αβ, αγ, ββ, and γγ.[3][6]
Three of these isoenzymes (all homodimers) are more commonly found in adult human cells than the others:

  • αα or non-neuronal enolase (NNE). Also known as enolase 1. Found in a variety of tissues, including liver, brain, kidney, spleen, adipose. It is present at some level in all normal human cells.
  • ββ or muscle-specific enolase (MSE). Also known as enolase 3. This enzyme is largely restricted to muscle where it is present at very high levels in muscle.
  • γγ or neuron-specific enolase (NSE). Also known as enolase 2. Expressed at very high levels in neurons and neural tissues, where it can account for as much as 3% of total soluble protein. It is expressed at much lower levels in most mammalian cells.

When present in the same cell, different isozymes readily form

heterodimers.[citation needed
]

Structure

Enolase is a member of the large

Glu20 of one subunit forms an ionic bond with Arg414 of the other subunit.[3] Each subunit has two distinct domains. The smaller N-terminal domain consists of three α-helices and four β-sheets.[3][6] The larger C-terminal domain starts with two β-sheets followed by two α-helices and ends with a barrel composed of alternating β-sheets and α-helices arranged so that the β-beta sheets are surrounded by the α-helices.[3][6]
The enzyme's compact, globular structure results from significant hydrophobic interactions between these two domains.

Enolase is a highly conserved enzyme with five active-site residues being especially important for activity. When compared to wild-type enolase, a mutant enolase that differs at either the Glu168, Glu211, Lys345, or Lys396 residue has an activity level that is cut by a factor of 105.[3] Also, changes affecting His159 leave the mutant with only 0.01% of its catalytic activity.[3] An integral part of enolase are two Mg2+ cofactors in the active site, which serve to stabilize negative charges in the substrate.[3][6]

Recently,

plasminogen, have brought interest to the enzymes' catalytic loops and their structural diversity.[7][8]

  • 3-D depiction of enolase dimer in antiparallel orientation. One dimer’s N-terminal Glu20 forms an ionic bond with the other’s C-terminal Arg414 to stabilize the enzyme’s quaternary structure.
    3-D depiction of enolase dimer in antiparallel orientation. One dimer’s N-terminal Glu20 forms an ionic bond with the other’s C-terminal Arg414 to stabilize the enzyme’s quaternary structure.
  • Active site of enolase in the middle of the C-terminal domain’s barrel. Depicted are two Mg2+ cofactors and five highly conserved residues imperative for proper catalytic function: His159, Glu168, Glu211, Lys345, Lys396.
    Active site of enolase in the middle of the C-terminal domain’s barrel. Depicted are two Mg2+ cofactors and five highly conserved residues imperative for proper catalytic function: His159, Glu168, Glu211, Lys345, Lys396.

Mechanism

Mechanism for conversion of 2PG to PEP.

Using isotopic probes, the overall mechanism for converting 2-PG to PEP is proposed to be an

E1cB elimination reaction involving a carbanion intermediate.[9] The following detailed mechanism is based on studies of crystal structure and kinetics.[3][10][11][12][13][14][15]
When the substrate, 2-phosphoglycerate, binds to α-enolase, its carboxyl group coordinates with two magnesium ion cofactors in the active site. This stabilizes the negative charge on the deprotonated oxygen while increasing the acidity of the alpha hydrogen. Enolase's Lys345 deprotonates the alpha hydrogen, and the resulting negative charge is stabilized by resonance to the carboxylate oxygen and by the magnesium ion cofactors. Following the creation of the carbanion intermediate, the hydroxide on C3 is eliminated as water with the help of Glu211, and PEP is formed.

Additionally, conformational changes occur within the enzyme that aid catalysis. In human α-enolase, the substrate is rotated into position upon binding to the enzyme due to interactions with the two catalytic magnesium ions,

Asp255 to Asn
256 allow Ser39 to coordinate with Mg2+ and close off the active site. In addition to coordination with the catalytic magnesium ions, the pKa of the substrate's alpha hydrogen is also lowered due to protonation of the phosphoryl group by His159 and its proximity to Arg374. Arg374 also causes Lys345 in the active site to become deprotonated, which primes Lys345 for its role in the mechanism.

Diagnostic uses

In recent medical experiments, enolase concentrations have been sampled in an attempt to diagnose certain conditions and their severity. For example, higher concentrations of enolase in

aldolase, pyruvate kinase, creatine kinase, and lactate dehydrogenase).[16]
The same study showed that the fastest rate of tumor growth occurred in patients with the highest levels of CSF enolase. Increased levels of enolase have also been identified in patients who have suffered a recent
cerebrovascular accident. It has been inferred that levels of CSF neuron-specific enolase, serum NSE, and creatine kinase (type BB) are indicative in the prognostic assessment of cardiac arrest victims.[17] Other studies have focused on the prognostic value of NSE values in cerebrovascular accident victims.[18]

Autoantibodies to alpha-enolase are associated with rheumatoid arthritis[19] and the rare syndrome called Hashimoto's encephalopathy.[20]

Inhibitors

Small-molecule inhibitors of enolase have been synthesized as chemical probes (substrate-analogues) of the catalytic mechanism of the enzyme and more recently, have been investigated as potential treatments for cancer and infectious diseases.

prokaryotic origin,[29] reflecting the strong evolutionary conservation of Enolase and the ancient origin of the glycolysis pathway. SF2312 is a chiral molecule with only the 3S-enantiomer showing Enolase inhibitory activity and biological activity against bacteria.[30] More recently, a derivative of SF2312, termed HEX, and a prodrug thereoff, POMHEX, were shown to exert anti-neoplastic activity against ENO1-deleted glioma in a pre-clinical intracranial orthotopic mouse model.[31] An allosteric binder, ENOblock[22] was initially described as an inhibitor of Enolase, but subsequently shown not to actually inhibit the enzyme, but rather, interfere with the Enolase in vitro enzymatic assay.[32] ENOblock was found to alter the cellular localization of enolase, influencing its secondary, non-glycolytic functions, such as transcription regulation.[33] Subsequent analysis using a commercial assay also indicated that ENOblock can inhibit enolase activity in biological contexts, such as cells and animal tissues.[33] Methylglyoxal has also been described as an inhibitor of human enolase.[34]

Active site transition state analogue Enolase inhibitors have been explored pre-clinically for the treatment of various microbial pathogens, as well as in precision oncology for tumors with 1p36 homozygous deletions, that lack ENO1.[31][35][36][37][38][39][40]

Fluoride is a known competitor of enolase's substrate 2-PG. Fluoride can form a complex with magnesium and phosphate, which binds in the active site instead of 2-PG.[4] One study found that fluoride could inhibit bacterial enolase in vitro.[41] The Enolase inhibitory activity of Fluoride anion may contribute to the anti-cavity effect of fluoride toothpaste, by limiting lactic acid (a product of glycolysis, which requires Enolase) production.[medical citation needed]

References

  1. PMID 9376357
    .
  2. ^ PDB: 2XSX​; Vollmar M, Krysztofinska E, Chaikuad A, Krojer T, Cocking R, Vondelft F, Bountra C, Arrowsmith CH, Weigelt J, Edwards A, Yue WW, Oppermann U (2010). "Crystal structure of human beta enolase ENOB". Protein Data Bank.
  3. ^
    S2CID 9191423
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  4. ^
    S2CID 86699159
    .
  5. ^ Lohman, K; Meyerhof, O (1934). "Über die enzymatische umwandlung von phosphoglyzerinsäure in brenztraubensäure und phosphorsäure" [Enzymatic transformation of phosphoglyceric acid into pyruvic and phosphoric acid]. Biochemische Zeitschrift (in German). 273: 60–72.
  6. ^
    PMID 1840492
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  7. PMID 15476816
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  8. S2CID 9976031
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  9. doi:10.1016/S0021-9258(18)62051-4
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  10. PMID 8634301
    .
  11. PMID 8994873
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  12. PMID 7703246
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  13. PMID 8049235
    .
  14. PMID 8605183
    .
  15. PMID 7547999
    .
  16. PMID 7334408
    .
  17. PMID 2742544
    .
  18. PMID 6747647
    .
  19. PMID 18821669
    .
  20. S2CID 43249019
    .
  21. PMID 6380574
    .
  22. ^
    PMID 23547795
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  23. PMID 1322695
    .
  24. ^
    PMID 8193144
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  25. PMID 17822439
    .
  26. PMID 22895339
    .
  27. ^ Watanabe H, Yoshida J, Tanaka E, Ito M, Miyadoh S, Shomura T (1986). "Studies on a new phosphonic acid antibiotic, SF-2312". Sci Rep Meiji Seika Kaisha. 25: 12–17.
  28. PMID 27723749
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  29. PMID 31745118
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  30. PMID 31324042
    .
  31. ^
    PMID 33230295
    .
  32. PMID 28030597
    .
  33. ^
    PMID 28272459
    .
  34. S2CID 85416928
    .
  35. PMID 34604112
    .
  36. PMID 34279224
    .
  37. S2CID 226052354
    .
  38. PMID 33086062
    .
  39. PMID 31745118
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  40. PMID 31324042
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  41. PMID 2318530
    .

Further reading

External links
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