Phosphopyruvate hydratase
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.
Reference Protein and Structure
- Sequence
-
P00924
(4.2.1.11)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Saccharomyces cerevisiae S288c (Baker's yeast)
- PDB
-
7enl
- MECHANISM OF ENOLASE: THE CRYSTAL STRUCTURE OF ENOLASE-MG2+-PHOSPHOGLYCERATE(SLASH) PHOSPHOENOLPYRUVATE COMPLEX AT 2.2-ANGSTROMS RESOLUTION
(2.2 Å)
- Catalytic CATH Domains
-
3.30.390.10
3.20.20.120
(see all for 7enl)
- Cofactors
- Magnesium(2+) (2) Metal MACiE
Enzyme Reaction (EC:4.2.1.11)
→
+
Alternative enzyme names: 14-3-2-protein, 2-phosphoglycerate dehydratase, 2-phosphoglycerate enolase, 2-phosphoglyceric dehydratase, Gamma-enolase, Enolase, Nervous-system specific enolase, Phosphoenolpyruvate hydratase, 2-phospho-D-glycerate hydro-lyase,
Enzyme Mechanism
Introduction
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 Roles
| UniProt | PDB* (7enl) | ||
| Glu212 | Glu211A | Acts as a general acid/base. Promotes elimination of the hydroxy group. | hydrogen bond donor, proton donor |
| His374 | His373A | His373 interacts with the Glu168/Glu211/H2O system, which promotes removal or addition of hydroxyl at carbon-3 of the substrate. | electrostatic stabiliser |
| Glu169 | Glu168A | Activates Glu211 to be the general acid/base by purturbating it's pKa. Part of the Glu211/G168/hydroxyl system. | repulsive charge-charge interaction, activator, increase acidity |
| Lys397 | Lys396A | Helps stabilise the negatively charged intermediates. | hydrogen bond donor, electrostatic stabiliser |
| Lys346 | Lys345A | Acts as a general acid/base. Abstracts the alpha proton from the substrate. | proton acceptor, hydrogen bond acceptor, electrostatic stabiliser |
| Ser40 | Ser39A | Forms part of the second metal binding site. | metal ligand |
| Asp247, Glu296, Asp321 | Asp246A, Glu295A, Asp320A | Forms part of the first metal binding site. | metal ligand |
| His160 | His159A | Polypeptide loop closure may keep a proton from His159 interacting with the substrate phosphoryl oxygen long enough to stabilise a carbanion intermediate. | electrostatic stabiliser |
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file;
y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain;
C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.
Chemical Components
assisted keto-enol tautomerisation, overall reactant used, intermediate formation, proton transfer, rate-determining step, overall product formed, dehydration, intermediate collapse, intermediate terminated, unimolecular elimination by the conjugate baseReferences
- Carmieli R et al. (2007), J Am Chem Soc, 129, 4240-4252. The Catalytic Mn2+Sites in the Enolase−Inhibitor Complex: Crystallography, Single-Crystal EPR, and DFT Calculations. DOI:10.1021/ja066124e. PMID:17367133.
- Leonard PG et al. (2016), Nat Chem Biol, 12, 1053-1058. SF2312 is a natural phosphonate inhibitor of enolase. DOI:10.1038/nchembio.2195. PMID:27723749.
- Mutlu O et al. (2016), Comput Biol Chem, 64, 134-144. Comprehensive structural analysis of the open and closed conformations of Theileria annulata enolase by molecular modelling and docking. DOI:10.1016/j.compbiolchem.2016.06.002. PMID:27343873.
- Wu Y et al. (2015), Acta Crystallogr D Biol Crystallogr, 71, 2457-2470. Octameric structure ofStaphylococcus aureusenolase in complex with phosphoenolpyruvate. DOI:10.1107/s1399004715018830. PMID:26627653.
- Baleva M et al. (2015), Int J Mol Sci, 16, 9354-9367. A Moonlighting Human Protein Is Involved in Mitochondrial Import of tRNA. DOI:10.3390/ijms16059354. PMID:25918939.
- Qin J et al. (2012), J Inorg Biochem, 111, 187-194. Structures of asymmetric complexes of human neuron specific enolase with resolved substrate and product and an analogous complex with two inhibitors indicate subunit interaction and inhibitor cooperativity. DOI:10.1016/j.jinorgbio.2012.02.011. PMID:22437160.
- Sims PA et al. (2003), Biochemistry, 42, 8298-8306. Reverse Protonation Is the Key to General Acid−Base Catalysis in Enolase†,‡. DOI:10.1021/bi0346345. PMID:12846578.
- Brewer JM et al. (2003), J Protein Chem, 22, 353-361. Enzymatic function of loop movement in enolase: preparation and some properties of H159N, H159A, H159F, and N207A enolases. PMID:13678299.
- Poyner RR et al. (2001), Biochemistry, 40, 8009-8017. Role of metal ions in catalysis by enolase: an ordered kinetic mechanism for a single substrate enzyme. PMID:11434770.
- Brewer JM et al. (2000), Biochem Biophys Res Commun, 276, 1199-1202. The H159A Mutant of Yeast Enolase 1 Has Significant Activity. DOI:10.1006/bbrc.2000.3618. PMID:11027610.
- Vinarov DA et al. (1998), Biochemistry, 37, 15238-15246. pH Dependence of the Reaction Catalyzed by Yeast Mg−Enolase†. DOI:10.1021/bi981047o. PMID:9790688.
- Brewer JM et al. (1997), Biochim Biophys Acta, 1340, 88-96. Effect of site-directed mutagenesis of His373 of yeast enolase on some of its physical and enzymatic properties. DOI:10.1016/s0167-4838(97)00029-0. PMID:9217018.
- Larsen TM et al. (1996), Biochemistry, 35, 4349-4358. 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†,‡. DOI:10.1021/bi952859c. PMID:8605183.
- Poyner RR et al. (1996), Biochemistry, 35, 1692-1699. Toward Identification of Acid/Base Catalysts in the Active Site of Enolase: Comparison of the Properties of K345A, E168Q, and E211Q Variants†. DOI:10.1021/bi952186y. PMID:8634301.
- Reed GH et al. (1996), Curr Opin Struct Biol, 6, 736-743. Structural and mechanistic studies of enolase. PMID:8994873.
- Zhang E et al. (1994), Biochemistry, 33, 6295-6300. Catalytic metal ion binding in enolase: the crystal structure of an enolase-Mn2+-phosphonoacetohydroxamate complex at 2.4-A resolution. DOI:10.2210/pdb1els/pdb. PMID:8193144.
- Lebioda L et al. (1991), Biochemistry, 30, 2817-2822. Mechanism of enolase: the crystal structure of enolase-magnesium-2-phosphoglycerate/phosphoenolpyruvate complex at 2.2-.ANG. resolution. DOI:10.1021/bi00225a012. PMID:2007120.
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.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Glu211A | hydrogen bond donor |
| Glu168A | repulsive charge-charge interaction, activator, increase acidity |
| Lys396A | hydrogen bond donor, electrostatic stabiliser |
| Lys345A | hydrogen bond acceptor |
| Ser39A | metal ligand |
| Asp320A | metal ligand |
| Glu295A | metal ligand |
| Asp246A | metal ligand |
| His159A | electrostatic stabiliser |
| His373A | electrostatic stabiliser |
| Lys345A | proton acceptor |
Chemical Components
assisted keto-enol tautomerisation, overall reactant used, intermediate formation, proton transfer, rate-determining step
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.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Ser39A | metal ligand |
| Asp320A | metal ligand |
| Glu295A | metal ligand |
| Asp246A | metal ligand |
| Lys345A | electrostatic stabiliser |
| His159A | electrostatic stabiliser |
| Lys396A | electrostatic stabiliser |
| Glu168A | increase acidity |
| His373A | electrostatic stabiliser |
| Glu211A | proton donor |
Chemical Components
overall product formed, dehydration, intermediate collapse, intermediate terminated, proton transfer, ingold: unimolecular elimination by the conjugate base
Download: Introduction
Dehydration proceeds via an EC1B mechanism. Rate determining deprotonation of the substrate C-2 proton by the catalytic base leads to Sp2 hybridization of the C-2 atom, forming an enolate and then subsequent trans-elimination of the C-3 hydroxyl group is facilitated by the proton donor His 159 residue. The eliminated water molecule is incorporated into the coordination sphere of the catalytic divalent Mg ion.
Catalytic Residues Roles
| UniProt | PDB* (7enl) | ||
| Lys397 | Lys396A | The residue acts as a general base towards the C-2 atoms of the substrate, forming an enolate through a carbanion transition state. | proton shuttle (general acid/base) |
| Glu212, His374, Glu169, Lys346 | Glu211A, His373A, Glu168A, Lys345A | Act to stabilise the reactive intermediates and transition states formed during the course of the reaction. | hydrogen bond acceptor, electrostatic stabiliser |
| Ser40 | Ser39A | Forms part of the second metal ion binding site. | metal ligand |
| Asp247, Glu296, Asp321 | Asp246A, Glu295A, Asp320A | Forms part of the first metal binding site. | metal ligand |
| His160 | His159A | The residue acts as a general acid towards the departing hydroxyl group, facilitating its elimination. Electrostatic interactions with the catalytically essential divalent Mg ion tunes the pKa of the residue towards its function. | proton shuttle (general acid/base) |
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file;
y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain;
C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.
Chemical Components
References
- Vinarov DA et al. (1998), Biochemistry, 37, 15238-15246. pH Dependence of the Reaction Catalyzed by Yeast Mg−Enolase†. DOI:10.1021/bi981047o. PMID:9790688.
- Zhang E et al. (1994), Biochemistry, 33, 6295-6300. Catalytic metal ion binding in enolase: the crystal structure of an enolase-Mn2+-phosphonoacetohydroxamate complex at 2.4-A resolution. DOI:10.2210/pdb1els/pdb. PMID:8193144.
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| His159A | proton shuttle (general acid/base) |
| Ser39A | metal ligand |
| Lys396A | proton shuttle (general acid/base) |
| Asp246A | metal ligand |
| Glu295A | metal ligand |
| Asp320A | metal ligand |
| Lys345A | hydrogen bond donor |
| Glu168A | hydrogen bond acceptor |
| Glu211A | hydrogen bond acceptor |
| His373A | hydrogen bond donor |
| Glu168A | electrostatic stabiliser |
| Glu211A | electrostatic stabiliser |
| Lys345A | electrostatic stabiliser |
| His373A | electrostatic stabiliser |