Enolase

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. The chemical reaction catalyzed by enolase is:

2-phospho-D-glycerate phosphoenolpyruvate + H2O

phosphopyruvate hydratase
Yeast enolase dimer.[1]
Identifiers
EC number4.2.1.11
CAS number9014-08-8
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
Enolase, N-terminal domain
x-ray structure and catalytic mechanism of lobster enolase
Identifiers
SymbolEnolase_N
PfamPF03952
Pfam clanCL0227
InterProIPR020811
PROSITEPDOC00148
SCOP21els / SCOPe / SUPFAM
Enolase
Crystal structure of dimeric beta human enolase ENO3.[2]
Identifiers
SymbolEnolase
PfamPF00113
InterProIPR000941
PROSITEPDOC00148

Enolase 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.[3] The optimum pH for the human 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 type X.

Isozymes

In humans 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). 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.

Structure

Enolase is a member of the large enolase superfamily. It 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]

Recently, moonlighting functions of several enolases, such as interaction with 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.
  • 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, 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).[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 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.[17] Other studies have focused on the prognostic value of NSE values in cerebrovascular accident victims.[18]

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

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.[20][21] Most inhibitors have metal chelating properties and bind to enzyme by interactions with the structural Magnesium Atom Mg(A).[22][23] The most potent of these is phosphonoacetohydroxamate,[23] 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,[24] 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 tumor suppressor locus (synthetic lethality).[25] A natural product phosphonate antibiotic, SF2312 (CAS 107729-45-3), which is active against gram positive and negative bacteria especially under anaerobic conditions,[26] is a high potency inhibitor of Enolase 4zcw that binds in manner similar to phoshphonoacetohydroxamate 4za0.[27] SF2312 inhibits Enolase activity in both eukaryotic and prokaryotic origin,[28] 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.[29] 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.[30] An allosteric binder, ENOblock [21] 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. [31] ENOblock was found to alter the cellular localization of enolase, influencing its secondary, non-glycolytic functions, such as transcription regulation.[32] Subsequent analysis using a commercial assay also indicated that ENOblock can inhibit enolase activity in biological contexts, such as cells and animal tissues.[32] Methylglyoxal has also been described as an inhibitor of human enolase. [33]

Fluoride is a known competitor of enolase's substrate 2-PG. Fluoride can 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.[34]

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). "Crystal structure of human beta enolase ENOB". To be Published.
  3. Pancholi V (June 2001). "Multifunctional alpha-enolase: its role in diseases". Cellular and Molecular Life Sciences. 58 (7): 902–20. doi:10.1007/pl00000910. PMID 11497239. S2CID 9191423.
  4. Hoorn RK, Flikweert JP, Staal GE (November 1974). "Purification and properties of enolase of human erythrocytes". International Journal of Biochemistry. 5 (11–12): 845–852. doi:10.1016/0020-711X(74)90119-0. hdl:1874/18158.
  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. Peshavaria M, Day IN (April 1991). "Molecular structure of the human muscle-specific enolase gene (ENO3)". The Biochemical Journal. 275 (Pt 2): 427–33. doi:10.1042/bj2750427. PMC 1150071. PMID 1840492.
  7. Ehinger S, Schubert WD, Bergmann S, Hammerschmidt S, Heinz DW (October 2004). "Plasmin(ogen)-binding alpha-enolase from Streptococcus pneumoniae: crystal structure and evaluation of plasmin(ogen)-binding sites". Journal of Molecular Biology. 343 (4): 997–1005. doi:10.1016/j.jmb.2004.08.088. PMID 15476816.
  8. Raghunathan K, Harris PT, Spurbeck RR, Arvidson CG, Arvidson DN (June 2014). "Crystal structure of an efficacious gonococcal adherence inhibitor: an enolase from Lactobacillus gasseri". FEBS Letters. 588 (14): 2212–6. doi:10.1016/j.febslet.2014.05.020. PMID 24859038. S2CID 9976031.
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  11. 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.
  12. 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 A resolution". Biochemistry. 34 (13): 4325–30. doi:10.1021/bi00013a022. PMID 7703246.
  13. 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-A resolution". Biochemistry. 33 (31): 9333–42. doi:10.1021/bi00197a038. PMID 8049235.
  14. 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 A resolution". Biochemistry. 35 (14): 4349–58. doi:10.1021/bi952859c. PMID 8605183.
  15. 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.
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  21. Jung DW, Kim WH, Park SH, Lee J, Kim J, Su D, Ha HH, Chang YT, Williams DR. (2 April 2013). "A unique small molecule inhibitor of enolase clarifies its role in fundamental biological processes". ACS Chemical Biology. 8 (6): 1271–1282. doi:10.1021/cb300687k. PMID 23547795.CS1 maint: uses authors parameter (link)
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  23. Zhang E, Hatada M, Brewer JM, Lebioda L (May 1994). "Catalytic metal ion binding in enolase: the crystal structure of an enolase-Mn2+-phosphonoacetohydroxamate complex at 2.4-A resolution". Biochemistry. 33 (20): 6295–300. doi:10.1021/bi00186a032. PMID 8193144.
  24. de A S Navarro MV, Gomes Dias SM, Mello LV, da Silva Giotto MT, Gavalda S, Blonski C, Garratt RC, Rigden DJ (October 2007). "Structural flexibility in Trypanosoma brucei enolase revealed by X-ray crystallography and molecular dynamics". The FEBS Journal. 274 (19): 5077–89. doi:10.1111/j.1742-4658.2007.06027.x. PMID 17822439.CS1 maint: uses authors parameter (link)
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  30. Lin YH, Satani N, Hammoudi N, Yan VC, Barekatain Y, Khadka S, et al. (December 2020). "An enolase inhibitor for the targeted treatment of ENO1-deleted cancers". Nature Metabolism. 2 (12): 1413–1426. doi:10.1038/s42255-020-00313-3. PMC 7744354. PMID 33230295.
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