M-CSA Mechanism and Catalytic Site Atlas

2-dehydro-3-deoxy-phosphogluconate aldolase

2-ket-3-deoxy-phosphogluconate (KDPG) aldolase is a Class I aldolase of the glycolytic pathway. It catalyses the reversible cleavage of KDPG into the three carbon units pyruvate and glyceraldehyde-3-phosphate. The enzymatic aldol reaction is highly efficient, regioselective and shows high facial stereoselectivity. The mechanism shown here is the formation KDGP from pyruvate and glyceraldehyde-3-phosphate.

Reference Protein and Structure

Sequence: P0A955 (4.1.2.14, 4.1.3.16) (Sequence Homologues) (PDB Homologues)
Biological species: Escherichia coli K-12 (Bacteria)
PDB: 1fq0 - KDPG ALDOLASE FROM ESCHERICHIA COLI (2.1 Å)
Catalytic CATH Domains: 3.20.20.70 (see all for 1fq0)
Cofactors: Fadh2(2-) (1)

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Enzyme Reaction (EC:4.1.2.14)

water

CHEBI:15377

pyruvate

CHEBI:15361

D-glyceraldehyde 3-phosphate(2-)

CHEBI:59776

→

2-dehydro-3-deoxy-6-phosphonato-D-gluconate(3-)

CHEBI:57569

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: 2-keto-3-deoxy-6-phosphogluconate aldolase, 2-keto-3-deoxy-6-phosphogluconic aldolase, 2-keto-3-deoxygluconate-6-P-aldolase, 2-keto-3-deoxygluconate-6-phosphate aldolase, 2-oxo-3-deoxy-6-phosphogluconate aldolase, 6-phospho-2-keto-3-deoxygluconate aldolase, KDPG aldolase, ODPG aldolase, Phospho-2-keto-3-deoxygluconate aldolase, Phospho-2-keto-3-deoxygluconic aldolase, Phospho-2-dehydro-3-deoxygluconate aldolase, KDPG-aldolase, 2-dehydro-3-deoxy-D-gluconate-6-phosphate D-glyceraldehyde-3-phosphate-lyase, 2-dehydro-3-deoxy-D-gluconate-6-phosphate D-glyceraldehyde-3-phosphate-lyase (pyruvate-forming),

Enzyme Mechanism

Mechanism Proposal 1 ☆☆☆

Summary
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
Step 7
Step 8
Products
All Steps

Introduction

In the aldol condensation direction, Glu45 acts as a general base towards the protonated Lys133 through a structurally conserved water molecule. This activates the lysine residue towards nucleophilic attack the the pyruvate carbonyl, which with concomitant protonation of the hydroxyl group from Glu45 results in the elimination of water and formation of an imine intermediate. Glu45 initiates tautomerisation through proton transfer with the eliminated water, forming a carbinolamine. The glyceraldehyde-3-phosphate is then attacked by this intermediate at its carbonyl functionality, with simultaneous protonation from Glu45. This residue then acts as a base towards a hydrolytic water which attacks the imine, and initiates the collapse of the tetrahedral intermediate, releasing the free Lys133, Glu45, KDPG and reforming the conserved water molecule.

Catalytic Residues Roles

UniProt	PDB* (1fq0)
Arg49	Arg49A	The close proximity of the residue's positively charged side chain to the catalytic nucleophile Lys133 influences the residue's pKa, increasing its acidity and so the interaction enhances Lys133 nucleophilic character.	modifies pKa, electrostatic stabiliser
Lys133	Lys133A	The residue acts as a nucleophile towards the substrate carbonyl, forming a Schiff imine with elimination of water. This water then remains in the active site to facilitate proton relay between the substrate and Glu45. Its pKa is modified through electrostatic interaction with Arg49 and the general base Glu45 via a water molecule.	nucleofuge, nucleophile, proton acceptor, proton donor, electron pair acceptor, electron pair donor
Glu45	Glu45A	The residue acts as a general base within a proton relay with a water molecule to deprotonate Lys133, and therefore activate the residue to act as a nucleophile towards the substrate carbonyl. It also acts as a general acid towards the eliminated water. Because of the distance between the most likely substrate binding position and the residue, it is thought that most of its acid/base interactions are mediated through a structurally conserved water molecule.	proton acceptor, proton donor

*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

proton transfer, bimolecular nucleophilic addition, enzyme-substrate complex formation, overall reactant used, intermediate formation, schiff base formed, bimolecular elimination, assisted keto-enol tautomerisation, aldol addition, overall product formed, enzyme-substrate complex cleavage, intermediate terminated, intramolecular elimination

References

Fullerton SW et al. (2006), Bioorg Med Chem, 14, 3002-3010. Mechanism of the Class I KDPG aldolase. DOI:10.1016/j.bmc.2005.12.022. PMID:16403639.

Step 1. Glu45 deprotonates Lys133 via a water molecule, activating the lysine. The positively charged Arg 49 acts to increase the acidity of the protonated lysine.

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Catalytic Residues Roles

Residue	Roles
Arg49A	electrostatic stabiliser, modifies pKa
Lys133A	proton donor
Glu45A	proton acceptor

Chemical Components

proton transfer

Step 2. The activated Lys133 performs a nucleophilic attack on the pyruvate carbonyl.

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Catalytic Residues Roles

Residue	Roles
Lys133A	nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, overall reactant used, intermediate formation

Step 3. The concomitant protonation of the hydroxyl by Glu45 leads to water being eliminated and an imine intermediate being formed.

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Catalytic Residues Roles

Residue	Roles
Glu45A	proton donor
Lys133A	electron pair donor

Chemical Components

proton transfer, schiff base formed, ingold: bimolecular elimination

Step 4. Glu45 initiates tautomerisation via proton transfer with the eliminated water, forming a carbinolamine.

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Catalytic Residues Roles

Residue	Roles
Lys133A	electron pair acceptor
Glu45A	proton acceptor

Chemical Components

proton transfer, assisted keto-enol tautomerisation

Step 5. The glyceraldehyde-3-phosphate is then attacked by this intermediate at its carbonyl functionality in an aldol addition, with simultaneous protonation from Glu45 via the water molecule.

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Catalytic Residues Roles

Residue	Roles
Glu45A	proton donor
Lys133A	electron pair donor

Chemical Components

aldol addition, proton transfer, overall reactant used, schiff base formed

Step 6. A hydrolytic water then attacks the imine upon deprotonation by Glu45 forming a tetrahedral intermediate.

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Catalytic Residues Roles

Residue	Roles
Glu45A	proton acceptor
Lys133A	electron pair acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 7. The amine group of the intermediate accepts a proton from Glu45 via a water molecule, to create a better leaving group.

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Catalytic Residues Roles

Residue	Roles
Glu45A	proton donor
Lys133A	proton acceptor

Chemical Components

proton transfer

Step 8. The tetrahedral intermediate collapses releasing Lys133 and forming the product with concomitant deprotonation of the hydroxyl by Glu45.

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Catalytic Residues Roles

Residue	Roles
Glu45A	proton acceptor
Lys133A	nucleofuge

Chemical Components

overall product formed, enzyme-substrate complex cleavage, intermediate terminated, ingold: intramolecular elimination, proton transfer