Enolase

phosphopyruvate hydratase
Yeast enolase dimer.
Identifiers
EC number	4.2.1.11
CAS number	Template:CAS
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
Search PMC articles PubMed articles NCBI proteins

Enolase, N-terminal domain
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
Available protein structures: Pfam structures PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

Enolase
Crystal structure of dimeric beta human enolase ENO3.
Identifiers
Symbol	Enolase
Pfam	PF00113
InterPro	IPR000941
PROSITE	PDOC00148
Available protein structures: Pfam structures PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

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 $\rightleftharpoons$ phosphoenolpyruvate + H₂O

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. The optimum pH for the human enzyme is 6.5. Enolase is present in all tissues and organisms capable of glycolysis or fermentation. The enzyme was discovered by Lohmann and Meyerhof in 1934, and has since been isolated from a variety of sources including human muscle and erythrocytes. In humans, deficiency of ENO1 is linked to hereditary haemolytic anemia while ENO3 deficiency is linked to glycogen storage disease XIII.

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. 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. 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. Each subunit has two distinct domains. The smaller N-terminal domain consists of three α-helices and four β-sheets. 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. 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. Also, changes affecting His¹⁵⁹ leave the mutant with only 0.01% of its catalytic activity. An integral part of enolase are two Mg²⁺ cofactors in the active site, which serve to stabilize negative charges in the substrate.

Recently, moonlighting functions of several enolases, such as interaction with plasminogen, have brought interest to the enzymes' catalytic loops and their structural diversity.

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.

Structural studies

As of late 2007, 27 structures have been solved for this enzyme, with PDB accession codes 1E9I, 1EBG, 1EBH, 1ELS, 1IYX, 1L8P, 1NEL, 1OEP, 1ONE, 1P43, 1P48, 1PDY, 1PDZ, 1TE6, 1W6T, 2AKM, 2AKZ, 2AL1, 2AL2, 2FYM, 2ONE, 2PA6, 3ENL, 4ENL, 5ENL, 6ENL, and 7ENL.

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. The following detailed mechanism is based on studies of crystal structure and kinetics. 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). 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. Other studies have focused on the prognostic value of NSE values in cerebrovascular accident victims.

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

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.

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. As such, drinking fluoridated water provides fluoride at a level that inhibits oral bacteria enolase activity. Disruption of the bacteria’s glycolytic pathway - and, thus, its normal metabolic functioning - prevents dental caries from forming.

References

• Boyer, P.D., Lardy, H. and Myrback, K. (Eds.), The Enzymes, 2nd ed., vol. 5, Academic Press, New York, 1961, p. 471-494.

External links

• Enolase at the US National Library of Medicine Medical Subject Headings (MeSH)