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| Enolase |
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Enolase, also known as phosphopyruvate dehydratase, is a metalloenzyme responsible for the catalysis 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.[1] The optimum pH for this enzyme is 6.5.[2] 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.[2] There are three subunits of enolase, α, β, and γ, which can combine to form five different isoenzymes: αα, αβ, αγ, ββ, and γγ.[1],[3] Three of these isoenzymes 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.
[edit] Structure
Enolase has a molecular weight of 82,000-100,000 Daltons depending on the isoform.[1],[2] 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.[1] Each subunit has two distinct domains. The smaller N-terminal domain consists of three α-helices and four β-sheets.[1],[3] The larger C-terminal domain starts with two β-sheets followed by two α-helices and ends with a barrel comprised of alternating β-sheets and α-helices arranged so that the β-beta sheets are surrounded by the α-helices.[1],[3] 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.[1] Also, changes affecting His159 leave the mutant with only 0.01% of its catalytic activity.[1] An integral part of enolase are two Mg2+ cofactors in the active site, which serve to stabilize negative charges in the substrate.[1],[3]
3-D depiction of enolase dimer in antiparallel orientation.
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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.
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[edit] 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 Elcb elimination reaction involving a carbanion intermediate.[4] The following detailed mechanism is based on studies of crystal structure and kinetics.[1],[5],[6],[7],[8],[8],[10] 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.
[edit] 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).[11] 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.[12] Other studies have focused on the prognostic value of NSE values in cerebrovascular accident victims.[13]
[edit] 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.[2] As such, drinking water fluoridation provides fluoride at a level which 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.[14],[15]
[edit] References
- Pancholi V. Multifunctional a-enolase: its role in diseases. Cell Mol Life Sci (2001) 58:902-920.
- Hoorn RK, Flickweert JP and Staal GE. Purification and properties of enolase of human erythroctyes. Int J Biochem (1974) 5:845-852.
- Peshavaria M and Day IN. Molecular structure of the human muscle-specific enolase gene (EN03). Biochemistry (1991) 275:427-433.
- Dinovo EC and Boyer PD. Isotopic probes of the enolase reaction mechanism. J Biol Chem (1971) 240:4586-4593.
- Poyner RR, Laughlin LT, Sowa GA and Reed GH. Toward identification of acid/base catalysts in the active site of enolase: Comparison of the properties of K345A, E168Q and E211Q variants. Biochemistry (1996) 35:1692–1699.
- Reed GH, Poyner RR, Larsen TM, Wedekind JE and Rayment I. Structural and mechanistic studies of enolase. Curr Opin Struct Biol (1996) 6:736–743
- Wedekind JE, Reed GH and Rayment I. 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 (1995) 34:4325–4330.
- Wedekind JE, Poyner RR, Reed GH and Rayment I. 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 (1994) 33:9333–9342.
- Larsen TM, Wedekind JE, Rayment I and Reed GH. 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 (1996) 35:4349–4358.
- Duquerroy S, Camus C and Janin J. X-ray structure and catalytic mechanism of lobster enolase. Biochemistry (1995) 34:12513–12523.
- Royds JA, Timperley WR and Taylor CB. Levels of enolase and other enzymes in the cerebrospinal fluid as indices of pathological change. J Neurol Neurosurg Pyschiatry (1981) 44:1129-1135.
- Roine RO, Somer H, Kaste M, Viinikka L and Karonen SL. Neurological outcome after out-of-hospital cardiac arrest. Prediction by cerebrospinal fluid enzyme analysis. Arch Neurol (1989) 46:753-756.
- Hay E, Royds JA, Davies-Jones GA, Lewtas NA, Timperley WR and Taylor CB. Cerebrospinal fluid enolase in stroke. J Neurol Neurosurg Psychiatry (1984) 47:724-729.
- Centers For Disease Control And Prevention, comp. Populations Receiving Optimally Fluoridated Public Drinking Water --- United States, 2000 Morbidity and Mortality Weekly Report (2002) 51: 144-147.
- Hüther, F-J, N. Psarros, H. Duschner. Isolation, characterization, and inhibition kinetics of enolase from Streptococcus rattus FA-1. Infection and Immunity (1990) 58:1043-1047.
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