Enzyme - Phosphopyruvate hydratase

Alternative Name(s)
  • 2-phosphoglycerate dehydratase.
  • Enolase.

Catalytic Activity

(2R)-2-phosphoglycerate = H2O + phosphoenolpyruvate



Reaction Mechanism

    Yeast enolase (2-phospho-D-glycerate hydrolase) is a metalloenzyme which catalyses the reversible dehydration of D-2-phos-phoglycerate (PGA) to phosphoenolpyruvate (PEP). The enzyme has an absolute requirement for the presence of a divalent cation, as is characteristic of the enolase family.

    Mitochondrial targeting of tRK1 in yeast is achieved by the successive actions of enolase 2 and the precursor of the mitochondrial lysyl-tRNA synthetase (preMSK). At the mitochondrial outer membrane, preMSK takes over enolase to start the import process properly; A fraction of the canonical tRNA L-form tRK1 pool is deviated from the cytosolic translation process by the enolase 2, which favours the tRNA conformational change leading to the formation of the F-form.

    This enzyme catalyses the inter-conversion of 2-PGA and PEP in a reversible manner. In the dehydration direction (shown here) both Lys345 and Glu211 (the catalytic acid/base pair) are neutral in charge [PMID:8634301, PMID:12846578]. At the end of the dehydration reaction, the residues are in the correct protonation state to perform the hydration reaction, i.e. Lys345 is positively charged and Glu211 negatively charged. It is assumed that at physiological pH that both protonation states of the enzyme coexist in reasonable proportions [PMID:12846578].
    Catalytic Residues
    AA Uniprot Uniprot Resid PDB PDB Resid
    Glu P00924 212 7enl 211
    His P00924 374 7enl 373
    Glu P00924 169 7enl 168
    Lys P00924 397 7enl 396
    Lys P00924 346 7enl 345
    Ser P00924 40 7enl 39
    Asp P00924 247 7enl 246
    Glu P00924 296 7enl 295
    Asp P00924 321 7enl 320
    His P00924 160 7enl 159
    Step Components

    intermediate formation, intermediate terminated, assisted keto-enol tautomerisation, rate-determining step, proton transfer, dehydration, overall reactant used, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed

    Step 1.

    Neutral Lys345 abstracts the proton alpha to the carbonyl group forming an oxyanion intermediate, which is stabilised by Lys396 and Mg(II) cations.

    Step 2.

    The oxyanion collapses, with concomitant double bond rearrangement, resulting in the elimination of the hydroxyl group, which abstracts a proton from the neutral Glu211.


    The products of the reaction.

Reaction Parameters

  • Kinetic Parameters
    Organism KM Value [mM] Substrate Comment
    Klebsiella pneumoniae 23.5 phosphoenolpyruvate in 50 mM imidazole-HCl buffer, pH 7.8, with 1 mM MgSO4, 0.4 M KCl
    Homo sapiens 48.5 phosphoenolpyruvate in 50 mM imidazole-HCl buffer, pH 6.8, with 3 mM MgSO4, 0.4 M KCl
    Saccharolobus solfataricus 58.44 phosphoenolpyruvate 65°C, pH not specified in the publication
    Theileria annulata 350 2-phospho-D-glycerate 1.5 mM MgCl2 in 50 mM Tris/HCl (pH 7.4), at 25°C
    Plasmodium falciparum 520 2-phospho-D-glycerate recombinant, dimeric enzyme
  • Temperature
    Organism Temperature Range Comment
    Bacillus anthracis 25 - 45 at pH 6.8, recombinant protein
    Clostridioides difficile 25 - 80 80°C: 27% of maximal activity
    Pyrococcus furiosus 40 - 90 no activity at 25°C, activity increases from 40°C to 90°C, the highest assay temperature used
  • pH
    Organism pH Range Comment
    Brucella abortus 5 - 10 activity range, recombinant enzyme
    Plasmodium falciparum 5 - 5.5 pH-dependent dissociation reveals that protonation of groups at the intersubunit interface is responsible for dissociation
    Macaca mulatta 6 - 9 pH 6.0: about 35% of maximal activity, pH 9.0: about 30% of maximal activity
    Trichinella spiralis 6 - 7.5
    Leuconostoc mesenteroides 6 - 8.3 catalytic activity above and below pH 6.8 remains constant at 30% of maximal activity

Associated Proteins

Protein name Organism
Enolase-related protein 3 Baker's yeast
Enolase 4 Mouse
Enolase-related protein 2 Baker's yeast
Enolase 1-2 Fission yeast
Gamma-enolase Human