Enolase

Enolase, also known as phosphopyruvate dehydratase, 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 can also catalyze the reverse reaction, depending on environmental concentrations of substrates. The optimum pH for this 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.

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. 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. Also known as enolase 1
 * ββ or muscle specific enolase (MSE). Also known as enolase 3.
 * γγ or neuron-specific enolase (NSE). Also known as enolase 2.

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.

Mechanism
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.

Fluoride Inhibition
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 without harming humans. Disruption of the bacteria’s glycolytic pathway - and, thus, its normal metabolic functioning - prevents dental caries from forming.