page_title: Enolase
section_title: Mechanism
hierarchical_section_title: Enolase / Mechanism
caption_attribution_description: English: Tram Nguyen &amp; Katelyn Thompson; Mechanism of enolase converting 2-phosphoglycerate to phosphoenolpyruvate, illustrated via ChemDraw 10.0.
context_page_description: Enolase, also known as phosphopyruvate hydratase, is a metalloenzyme responsible for the catalysis of the conversion of 2-phosphoglycerate to phosphoenolpyruvate, the ninth and penultimate step of glycolysis. The chemical reaction catalyzed by enolase is:
  2-phospho-D-glycerate 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.
  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 type X.
context_section_description: 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.
caption: Mechanism for conversion of 2PG to PEP.
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