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108  structures 5866  species 2  interactions 7258  sequences 14  architectures

Family: Enolase_C (PF00113)

Summary: Enolase, C-terminal TIM barrel domain

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Enolase Edit Wikipedia article

phosphopyruvate hydratase
Enolase 2ONE wpmp.png
Yeast enolase dimer.[1]
Identifiers
EC number 4.2.1.11
CAS number 9014-08-8
Databases
IntEnz IntEnz view
BRENDA BRENDA entry
ExPASy NiceZyme view
KEGG KEGG entry
MetaCyc metabolic pathway
PRIAM profile
PDB structures RCSB PDB PDBe PDBsum
Gene Ontology AmiGO / EGO
Enolase, N-terminal domain
PDB 1pdz EBI.jpg
x-ray structure and catalytic mechanism of lobster enolase
Identifiers
Symbol Enolase_N
Pfam PF03952
Pfam clan CL0227
InterPro IPR020811
PROSITE PDOC00148
SCOP 1els
SUPERFAMILY 1els
Enolase
2XSX.pdb.png
Crystal structure of dimeric beta human enolase ENO3.[2]
Identifiers
Symbol Enolase
Pfam PF00113
InterPro IPR000941
PROSITE PDOC00148

Enolase, also known as phosphopyruvate hydratase, is a metalloenzyme responsible for the catalysis of the conversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP), the ninth and penultimate step of glycolysis. Enolase belongs to the class Lyase. Enolase can also catalyze the reverse reaction, depending on environmental concentrations of substrates.[3] The optimum pH for this enzyme is 6.5.[4] Enolase is present in all tissues and organisms capable of glycolysis or fermentation. The enzyme was discovered by Lohmann and Meyerhof in 1934,[5] and has since been isolated from a variety of sources including human muscle and erythrocytes.[4] In humans, deficiency of ENO1 is linked to hereditary haemolytic anemia while ENO3 deficiency is linked to glycogen storage disease XIII.

Isozymes

There are three subunits of enolase, α, β, and γ, each encoded by a separate gene that can combine to form five different 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), which is found in a variety of tissues, including liver, brain, kidney, spleen, adipose. It is present at some level in all normal human cells. Also known as enolase 1
  • 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.

Structure

Enolase has a molecular weight of 82,000-100,000 Daltons depending on the isoform.[3][4] In human alpha enolase, the two subunits are antiparallel in orientation so that 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]

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

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.[7] The following detailed mechanism is based on studies of crystal structure and kinetics.[3][8][9][10][11][12][13] 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, Gln167, and Lys396. Movements of loops Ser36 to His43, Ser158 to Gly162, and Asp255 to Asn256 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 cerebrospinal fluid more strongly correlated to low-grade astrocytoma than did other enzymes tested (aldolase, pyruvate kinase, creatine kinase, and lactate dehydrogenase).[14] 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 myocardial infarction or 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.[15] Other studies have focused on the prognostic value of NSE values in cerebrovascular accident victims.[16]

Autoantibodies to alpha-enolase are associated with the rare syndrome called Hashimoto's encephalopathy.[17]

Inhibitors of Enolase

Small-molecule inhibitors of enolase have been synthesized as chemical probes of the catalytic mechanism of the enzyme. The most potent of these is phosphonoacetohydroxamate, which in its unprotonated form has pM affinity for the enzyme. It has structural similarity to the presumed catalytic intermediate, between PEP and 2-PG. Attempts have been made to use this inhibitor as an anti-trypanosome drug, and more recently, as an anti-cancer agent, specifically, in glioblastoma that are enolase-deficient due to homozygous deletion of the ENO1 gene as part of the 1p36 locus.[citation needed]


Fluoride is a known competitor of enolase’s substrate 2-PG. The fluoride is part of a complex with magnesium and phosphate, which binds in the active site instead of 2-PG.[4] As such, drinking fluoridated water provides fluoride at a level that inhibits oral bacteria enolase activity without harming humans. Disruption of the bacteria’s glycolytic pathway - and, thus, its normal metabolic functioning - prevents dental caries from forming.[18][19]

References

  1. ^ PDB 2ONE; Zhang E, Brewer JM, Minor W, Carreira LA, Lebioda L (October 1997). "Mechanism of enolase: the crystal structure of asymmetric dimer enolase-2-phospho-D-glycerate/enolase-phosphoenolpyruvate at 2.0 A resolution". Biochemistry 36 (41): 12526–34. doi:10.1021/bi9712450. 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). To be published. 
  3. ^ a b c d e f g h i j Pancholi V (June 2001). "Multifunctional α-enolase: its role in diseases". Cell Mol Life Sci. 58 (7): 902–20. doi:10.1007/PL00000910. PMID 11497239. 
  4. ^ a b c d Hoorn RK, Flickweert JP, Staal GE (1974). "Purification and properties of enolase of human erythroctyes". Int J Biochem 5 (11–12): 845–52. doi:10.1016/0020-711X(74)90119-0. 
  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). Biochem Z 273, 60–72.
  6. ^ a b c d Peshavaria M, Day IN (April 1991). "Molecular structure of the human muscle-specific enolase gene (ENO3)". Biochem J. 275 (Pt 2): 427–33. PMC 1150071. PMID 1840492. 
  7. ^ Dinovo EC, Boyer PD (1971). "Isotopic probes of the enolase reaction mechanism". J Biol Chem 240: 4586–93. 
  8. ^ Poyner RR, Laughlin LT, Sowa GA, Reed GH (February 1996). "Toward identification of acid/base catalysts in the active site of enolase: comparison of the properties of K345A, E168Q, and E211Q variants". Biochemistry 35 (5): 1692–9. doi:10.1021/bi952186y. PMID 8634301. 
  9. ^ Reed GH, Poyner RR, Larsen TM, Wedekind JE, Rayment I (December 1996). "Structural and mechanistic studies of enolase". Current Opinion in Structural Biology 6 (6): 736–43. doi:10.1016/S0959-440X(96)80002-9. PMID 8994873. 
  10. ^ Wedekind JE, Reed GH, Rayment I (April 1995). "Octahedral coordination at the high-affinity metal site in enolase: crystallographic analysis of the MgII—enzyme complex from yeast at 1.9 Å resolution". Biochemistry 34 (13): 4325–30. doi:10.1021/bi00013a022. PMID 7703246. 
  11. ^ Wedekind JE, Poyner RR, Reed GH, Rayment I (August 1994). "Chelation of serine 39 to Mg2+ latches a gate at the active site of enolase: structure of the bis(Mg2+) complex of yeast enolase and the intermediate analog phosphonoacetohydroxamate at 2.1-Å resolution". Biochemistry 33 (31): 9333–42. doi:10.1021/bi00197a038. PMID 8049235. 
  12. ^ Larsen TM, Wedekind JE, Rayment I, Reed GH (April 1996). "A carboxylate oxygen of the substrate bridges the magnesium ions at the active site of enolase: structure of the yeast enzyme complexed with the equilibrium mixture of 2-phosphoglycerate and phosphoenolpyruvate at 1.8 Å resolution". Biochemistry 35 (14): 4349–58. doi:10.1021/bi952859c. PMID 8605183. 
  13. ^ Duquerroy S, Camus C, Janin J (October 1995). "X-ray structure and catalytic mechanism of lobster enolase". Biochemistry 34 (39): 12513–23. doi:10.1021/bi00039a005. PMID 7547999. 
  14. ^ Royds JA, Timperley WR, Taylor CB (December 1981). "Levels of enolase and other enzymes in the cerebrospinal fluid as indices of pathological change". J Neurol Neurosurg Psychiatr. 44 (12): 1129–35. doi:10.1136/jnnp.44.12.1129. PMC 491233. PMID 7334408. 
  15. ^ Roine RO, Somer H, Kaste M, Viinikka L, Karonen SL (July 1989). "Neurological outcome after out-of-hospital cardiac arrest. Prediction by cerebrospinal fluid enzyme analysis". Arch Neurol. 46 (7): 753–6. doi:10.1001/archneur.1989.00520430047015. PMID 2742544. 
  16. ^ Hay E, Royds JA, Davies-Jones GA, Lewtas NA, Timperley WR, Taylor CB (July 1984). "Cerebrospinal fluid enolase in stroke". J Neurol Neurosurg Psychiatr. 47 (7): 724–9. doi:10.1136/jnnp.47.7.724. PMC 1027902. PMID 6747647. 
  17. ^ Fujii A, Yoneda M, Ito T, Yamamura O, Satomi S, Higa H, Kimura A, Suzuki M, Yamashita M, Yuasa T, Suzuki H, Kuriyama M (May 2005). "Autoantibodies against the amino terminal of alpha-enolase are a useful diagnostic marker of Hashimoto's encephalopathy". J. Neuroimmunol. 162 (1–2): 130–6. doi:10.1016/j.jneuroim.2005.02.004. PMID 15833368. 
  18. ^ Centers For Disease Control; Centers for Disease Control and Prevention (CDC) (February 2002). "Populations receiving optimally fluoridated public drinking water—United States, 2000". MMWR Morb Mortal Wkly Rep. 51 (7): 144–7. PMID 11905481. 
  19. ^ Hüther FJ, Psarros N, Duschner H (1 April 1990). "Isolation, characterization, and inhibition kinetics of enolase from Streptococcus rattus FA-1". Infect Immun. 58 (4): 1043–7. PMC 258580. PMID 2318530. 

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Enolase, C-terminal TIM barrel domain Provide feedback

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External database links

This tab holds annotation information from the InterPro database.

InterPro entry IPR020810

Enolase (2-phospho-D-glycerate hydrolase) is an essential glycolytic enzyme that catalyses the interconversion of 2-phosphoglycerate and phosphoenolpyruvate [PUBMED:1859865, PUBMED:1840492]. In vertebrates, there are 3 different, tissue-specific isoenzymes, designated alpha, beta and gamma. Alpha is present in most tissues, beta is localised in muscle tissue, and gamma is found only in nervous tissue. The functional enzyme exists as a dimer of any 2 isoforms. In immature organs and in adult liver, it is usually an alpha homodimer, in adult skeletal muscle, a beta homodimer, and in adult neurons, a gamma homodimer. In developing muscle, it is usually an alpha/beta heterodimer, and in the developing nervous system, an alpha/gamma heterodimer [PUBMED:3390159]. The tissue specific forms display minor kinetic differences. Tau-crystallin, one of the major lens proteins in some fish, reptiles and birds, has been shown [PUBMED:3589669] to be evolutionary related to enolase.

Neuron-specific enolase is released in a variety of neurological diseases, such as multiple sclerosis and after seizures or acute stroke. Several tumour cells have also been found positive for neuron-specific enolase. Beta-enolase deficiency is associated with glycogenosis type XIII defect.

Gene Ontology

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Domain organisation

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Pfam Clan

This family is a member of clan Enolase_TIM (CL0256), which has the following description:

This clan contains enzymes which adopt a TIM barrel fold.

The clan contains the following 5 members:

DUF849 Enolase_C MAAL_C MR_MLE MR_MLE_C

Alignments

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Full
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Representative proteomes NCBI
(5284)
Meta
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RP15
(546)
RP35
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RP55
(1367)
RP75
(1661)
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  Seed
(11)
Full
(7258)
Representative proteomes NCBI
(5284)
Meta
(3499)
RP15
(546)
RP35
(1007)
RP55
(1367)
RP75
(1661)
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Curation and family details

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Seed source: Prosite
Previous IDs: enolase;
Type: Domain
Author: Sonnhammer ELL
Number in seed: 11
Number in full: 7258
Average length of the domain: 256.00 aa
Average identity of full alignment: 52 %
Average coverage of the sequence by the domain: 66.97 %

HMM information View help on HMM parameters

HMM build commands:
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 23193494 -E 1000 --cpu 4 HMM pfamseq
Model details:
Parameter Sequence Domain
Gathering cut-off 21.2 21.2
Trusted cut-off 21.2 21.2
Noise cut-off 21.1 21.1
Model length: 296
Family (HMM) version: 17
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Species distribution

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Interactions

There are 2 interactions for this family. More...

Enolase_N Enolase_C

Structures

For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the Enolase_C domain has been found. There are 108 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein seqence.

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