Fructose-bisphosphate aldolase (Class I)

 

Fructose-1,6-bisphosphate muscle aldolase is an essential glycolytic enzyme that catalyses reversible carbon-carbon bond formation by cleaving fructose 1,6-bisphosphate (FBP) to yield dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (G3P), or in the other direction the condensation of DHAP and G3P to form FBP.

 

Reference Protein and Structure

Sequence
P00883 UniProt (4.1.2.13) IPR000741 (Sequence Homologues) (PDB Homologues)
Biological species
Oryctolagus cuniculus (rabbit) Uniprot
PDB
2qut - Dihydroxyacetone phosphate enamine intermediate in fructose-1,6-bisphosphate aldolase from rabbit muscle (1.88 Å) PDBe PDBsum 2qut
Catalytic CATH Domains
3.20.20.70 CATHdb (see all for 2qut)
Click To Show Structure

Enzyme Reaction (EC:4.1.2.13)

D-glyceraldehyde 3-phosphate(2-)
CHEBI:59776ChEBI
+
glycerone phosphate(2-)
CHEBI:57642ChEBI
→
D-fructofuranose 1,6-bisphosphate(4-)
CHEBI:49299ChEBI
Alternative enzyme names: 1,6-diphosphofructose aldolase, SMALDO, Aldolase, Diphosphofructose aldolase, Fructoaldolase, Fructose 1,6-diphosphate aldolase, Fructose 1-monophosphate aldolase, Fructose 1-phosphate aldolase, Fructose diphosphate aldolase, Fructose-1,6-bisphosphate triosephosphate-lyase, Ketose 1-phosphate aldolase, Phosphofructoaldolase, Zymohexase, D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase,

Enzyme Mechanism

Introduction

The catalytic mechanism of archaic fructose-1,6-bisphosphate aldolase proceeds after binding and ring-opening of the substrate. Lys 229 attacks the C2-carbonyl carbon nucleophilically to form a carbinolamine using general acid catalysis as well. General acid catalysis by Glu187 (Tyr146 is Archea) allows dehydration to form the imine or hydrolysis in the reverse direction - the C2 hydroxyl is protonated to leave as water to leave the Schiff-base or deprotonated for attack of the imine in the reverse. Proton abstraction by Asp 33 leads to C3-C4 bond cleavage to release glucose 3-phosphate and leave a carbanion/enamine which is protonated by Asp 33 to form the product imine. Glu187 activates a water for nucleophilic attack by general base catalysis which is followed by general base catalysis by Lys 229 to regenerate the active site and give release of dihydroxyacetone phosphate.

Assignment of the role of charged active site residues is complex as these residues can mediate proton transfers by general acid/base catalysis, stabilise or destabilise charges, and because of their proximity to each other are susceptible to electrostatic modification of their pKa charges.

Catalytic Residues Roles

UniProt PDB* (2qut)
Asp34 Asp33A As well as substrate binding and positioning, the negative charge of Asp33 is important for promoting the charged form of Lys146, ensuring stabilisation of intermediates and efficient proton transfers. hydrogen bond acceptor, electrostatic stabiliser
Lys147 Lys146A The charged form of Lys146 stabilises negative charges in the substrate and intermediates, and encourages the negatively charged form of Tyr363. hydrogen bond donor, electrostatic stabiliser
Glu188 Glu187A Involved in several key proton transfers. This residue is Tyr146 in Archea. hydrogen bond acceptor, hydrogen bond donor, polar interaction, proton donor, proton relay, proton acceptor, electrostatic stabiliser
Tyr364 Tyr363A Stereospecifically removes the C3 pro-S proton from the substrate. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Lys230 Lys229A Acts as the nucleophile in formation of the Schiff-base and activates the substrate and facilitates loss of the intermediate through general acid/base catalysis. covalently attached, hydrogen bond acceptor, hydrogen bond donor, nucleophile, polar interaction, proton donor, proton relay, proton acceptor, nucleofuge, electron pair acceptor, electron pair donor
Ser301 Ser300A Stabilises enamine intermediate. hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Glu190 Glu189A Activates a water molecule. activator, hydrogen bond acceptor, polar interaction, 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

bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used, proton transfer, proton relay, unimolecular elimination by the conjugate base, intermediate collapse, enzyme-substrate complex cleavage, schiff base formed, dehydration, assisted tautomerisation (not keto-enol), intramolecular nucleophilic addition, intermediate terminated, overall product formed, native state of enzyme regenerated, cyclisation

References

  1. St-Jean M et al. (2007), J Biol Chem, 282, 31028-31037. Stereospecific Proton Transfer by a Mobile Catalyst in Mammalian Fructose-1,6-bisphosphate Aldolase. DOI:10.1074/jbc.m704968200. PMID:17728250.
  2. Heron PW et al. (2017), J Biol Chem, 292, 19849-19860. Isomer activation controls stereospecificity of class I fructose-1,6-bisphosphate aldolases. DOI:10.1074/jbc.M117.811034. PMID:28972169.
  3. St-Jean M et al. (2009), Biochemistry, 48, 4528-4537. Charge Stabilization and Entropy Reduction of Central Lysine Residues in Fructose-Bisphosphate Aldolase. DOI:10.1021/bi8021558. PMID:19354220.
  4. St-Jean M et al. (2005), J Biol Chem, 280, 27262-27270. High Resolution Reaction Intermediates of Rabbit Muscle Fructose-1,6-bisphosphate Aldolase: SUBSTRATE CLEAVAGE AND INDUCED FIT. DOI:10.1074/jbc.m502413200. PMID:15870069.
  5. Lorentzen E et al. (2005), Biochemistry, 44, 4222-4229. Mechanism of the Schiff Base Forming Fructose-1,6-bisphosphate Aldolase:  Structural Analysis of Reaction Intermediates‡. DOI:10.1021/bi048192o. PMID:15766250.
  6. Lorentzen E et al. (2003), J Biol Chem, 278, 47253-47260. Crystal Structure of an Archaeal Class I Aldolase and the Evolution of (  )8 Barrel Proteins. DOI:10.1074/jbc.m305922200. PMID:12941964.
  7. Choi KH et al. (2001), Biochemistry, 40, 13868-13875. Snapshots of Catalysis:  the Structure of Fructose-1,6-(bis)phosphate Aldolase Covalently Bound to the Substrate Dihydroxyacetone Phosphate†,‡. DOI:10.1021/bi0114877. PMID:11705376.
  8. Littlechild JA et al. (1993), Trends Biochem Sci, 18, 36-39. A data-based reaction mechanism for type I fructose bisphosphate aldolase. DOI:10.1016/0968-0004(93)90048-r. PMID:8488556.

Step 1. Lys229 attacks the carbonyl carbon of D-glyceraldehyde 3- phosphate in a nucleophilic addition. First step in the formation of the Schiff base. Lttlechild et al. suggest that the Lys229 is uncharged at the start of the reaction [PMID:8488556]. St-Jean et al. suggest that a proton relay can occur through the Glu187 [PMID:17728250,PMID:15870069].

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Catalytic Residues Roles

Residue Roles
Lys229A polar interaction, hydrogen bond donor
Glu187A electrostatic stabiliser, hydrogen bond donor, polar interaction
Glu189A polar interaction, hydrogen bond acceptor, electrostatic stabiliser
Lys146A hydrogen bond donor, electrostatic stabiliser
Ser300A hydrogen bond acceptor, hydrogen bond donor
Asp33A hydrogen bond acceptor, electrostatic stabiliser
Lys229A nucleophile

Chemical Components

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

Step 2. The oxyanion formed deprotonates the bound Lys229.

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Catalytic Residues Roles

Residue Roles
Lys229A hydrogen bond donor
Glu187A electrostatic stabiliser, hydrogen bond donor, hydrogen bond acceptor, proton relay, polar interaction
Glu189A polar interaction, hydrogen bond acceptor, electrostatic stabiliser
Lys146A hydrogen bond donor, electrostatic stabiliser
Ser300A hydrogen bond acceptor, hydrogen bond donor
Asp33A hydrogen bond acceptor, electrostatic stabiliser
Glu187A proton donor
Lys229A proton donor
Glu187A proton acceptor

Chemical Components

proton transfer, intermediate formation, proton relay

Step 3. Lys229 initiates an elimination of water, which gains an extra proton from Glu187.

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Catalytic Residues Roles

Residue Roles
Lys229A covalently attached, hydrogen bond donor
Glu187A electrostatic stabiliser, hydrogen bond donor, polar interaction
Glu189A polar interaction, hydrogen bond acceptor, electrostatic stabiliser
Lys146A hydrogen bond donor, electrostatic stabiliser
Ser300A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Asp33A hydrogen bond acceptor, electrostatic stabiliser
Glu187A proton donor
Lys229A electron pair donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, intermediate formation, intermediate collapse, enzyme-substrate complex cleavage, schiff base formed, dehydration

Step 4. The phosphate of the bound intermediate deprotonates water, which in turn deprotonates Tyr363. The C-terminal tyrosine is highly mobile and the temporary binding of the C-terminus expels one of the catalytic waters from the active site. The tyrosine is activated by sequential proton transfer through a conserved water molecule that is hydrogen-bonded to the iminium phosphate dianion that acts as a conjugate base. The tyrosine phenate anion is stabilised by Lys146. [PMID:17728250]

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Catalytic Residues Roles

Residue Roles
Lys229A covalently attached, hydrogen bond donor
Glu187A electrostatic stabiliser, hydrogen bond acceptor, polar interaction
Glu189A polar interaction, hydrogen bond acceptor, electrostatic stabiliser, activator
Lys146A hydrogen bond donor, electrostatic stabiliser
Ser300A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Asp33A hydrogen bond acceptor, electrostatic stabiliser
Tyr363A hydrogen bond donor, proton donor

Chemical Components

proton transfer, intermediate formation, proton relay

Step 5. Tyr363 deprotonates the C1-H of the bound intermediate, initiating a double bond rearrangement that leaves Lys229 with a lone pair of electrons. Ser300 stabilises the enamine form. There is interconversion between the iminium and enamine forms of the covalently bound substrate [PMID:17728250].

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Catalytic Residues Roles

Residue Roles
Lys229A covalently attached, hydrogen bond donor
Glu187A electrostatic stabiliser, hydrogen bond acceptor, polar interaction
Glu189A polar interaction, hydrogen bond acceptor, electrostatic stabiliser, activator
Lys146A hydrogen bond donor, electrostatic stabiliser
Ser300A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Asp33A hydrogen bond acceptor, electrostatic stabiliser
Tyr363A hydrogen bond acceptor, proton acceptor
Lys229A electron pair acceptor

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol), intermediate formation

Step 6. Glu187 deprotonates water, which deprotonates a second water which deprotonates the phosphate of the bound intermediate. C-terminal expulsion allows rebinding of one of the catalytic waters. Ser300 stabilises the enamine form. There is interconversion between the iminium and enamine forms of the covalently bound substrate [PMID:17728250]. The enamine phosphate catalyses proton transfer through intervening water molecules to generate the acid form of Glu187.

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Catalytic Residues Roles

Residue Roles
Lys229A covalently attached, hydrogen bond donor
Glu187A electrostatic stabiliser, hydrogen bond acceptor, polar interaction
Glu189A polar interaction, hydrogen bond acceptor, electrostatic stabiliser, activator
Lys146A hydrogen bond donor, electrostatic stabiliser
Ser300A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Asp33A hydrogen bond acceptor, electrostatic stabiliser
Glu187A proton acceptor

Chemical Components

proton transfer, intermediate formation, proton relay

Step 7. Glycerone phosphate binds, displacing one of the water molecules. Lys229 initiates a nucleophilic addition of the bound intermediate to the glycerone phosphate. The formed oxyanion deprotonates Glu187.

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Catalytic Residues Roles

Residue Roles
Lys229A covalently attached, hydrogen bond donor
Glu187A electrostatic stabiliser, hydrogen bond donor, polar interaction
Glu189A polar interaction, hydrogen bond acceptor, electrostatic stabiliser, activator
Lys146A hydrogen bond donor, electrostatic stabiliser
Ser300A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Asp33A hydrogen bond acceptor, electrostatic stabiliser
Glu187A proton donor
Lys229A electron pair donor

Chemical Components

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

Step 8. Glu187 deprotonates water, which attacks the imine carbon of the covalenlty bound intermediate in a nucleophilic addition.

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Catalytic Residues Roles

Residue Roles
Lys229A covalently attached, hydrogen bond donor
Glu187A electrostatic stabiliser, hydrogen bond acceptor, polar interaction
Glu189A polar interaction, hydrogen bond acceptor, electrostatic stabiliser, activator
Lys146A hydrogen bond donor, electrostatic stabiliser
Ser300A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Asp33A hydrogen bond acceptor, electrostatic stabiliser
Glu187A proton acceptor
Lys229A electron pair acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, intermediate formation, enzyme-substrate complex formation

Step 9. Lys229 deprotonates the formed hydroxyl group, which initiates an elimination of the linear form of D-fructose 1,6-bisphosphate and neutral Lys229.

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Catalytic Residues Roles

Residue Roles
Lys229A covalently attached, hydrogen bond donor, hydrogen bond acceptor
Glu187A electrostatic stabiliser, hydrogen bond donor, hydrogen bond acceptor, polar interaction, proton relay
Glu189A polar interaction, hydrogen bond acceptor, electrostatic stabiliser, activator
Lys146A hydrogen bond donor, electrostatic stabiliser
Ser300A hydrogen bond acceptor, hydrogen bond donor
Asp33A hydrogen bond acceptor, electrostatic stabiliser
Lys229A proton acceptor
Glu187A proton donor
Lys229A nucleofuge
Glu187A proton acceptor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, intermediate collapse, enzyme-substrate complex cleavage, intermediate formation, proton relay

Step 10. There is evidence in archea that the ring opening/closing reaction is enzyme catalysed by the Lys229. Phosphate deprotonates hydroxyl, and the carbonyl oxygen re-protonates from Lys229, which in turn re-protonates from the phosphate. [PMID:15766250] thus catalysing the ring closure of D-fructose 1,6-bisphosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys229A hydrogen bond donor, hydrogen bond acceptor, proton relay
Glu187A electrostatic stabiliser, hydrogen bond donor, polar interaction
Glu189A polar interaction, hydrogen bond acceptor, electrostatic stabiliser, activator
Lys146A hydrogen bond donor, electrostatic stabiliser
Ser300A hydrogen bond acceptor, hydrogen bond donor
Asp33A hydrogen bond acceptor, electrostatic stabiliser
Lys229A proton donor, proton acceptor

Chemical Components

proton transfer, ingold: intramolecular nucleophilic addition, intermediate terminated, proton relay, overall product formed, native state of enzyme regenerated, cyclisation
Download: Image, Marvin File

Step 1. Lys229 attacks the carbonyl carbon of D-glyceraldehyde 3- phosphate in a nucleophilic addition. First step in the formation of the Schiff base. Lttlechild et al. suggest that the Lys229 is uncharged at the start of the reaction [PMID:8488556]. St-Jean et al. suggest that a proton relay can occur through the Glu187 [PMID:17728250,PMID:15870069].

Step 2. The oxyanion formed deprotonates the bound Lys229.

Step 3. Lys229 initiates an elimination of water, which gains an extra proton from Glu187.

Step 4. The phosphate of the bound intermediate deprotonates water, which in turn deprotonates Tyr363. The C-terminal tyrosine is highly mobile and the temporary binding of the C-terminus expels one of the catalytic waters from the active site. The tyrosine is activated by sequential proton transfer through a conserved water molecule that is hydrogen-bonded to the iminium phosphate dianion that acts as a conjugate base. The tyrosine phenate anion is stabilised by Lys146. [PMID:17728250]

Step 5. Tyr363 deprotonates the C1-H of the bound intermediate, initiating a double bond rearrangement that leaves Lys229 with a lone pair of electrons. Ser300 stabilises the enamine form. There is interconversion between the iminium and enamine forms of the covalently bound substrate [PMID:17728250].

Step 6. Glu187 deprotonates water, which deprotonates a second water which deprotonates the phosphate of the bound intermediate. C-terminal expulsion allows rebinding of one of the catalytic waters. Ser300 stabilises the enamine form. There is interconversion between the iminium and enamine forms of the covalently bound substrate [PMID:17728250]. The enamine phosphate catalyses proton transfer through intervening water molecules to generate the acid form of Glu187.

Step 7. Glycerone phosphate binds, displacing one of the water molecules. Lys229 initiates a nucleophilic addition of the bound intermediate to the glycerone phosphate. The formed oxyanion deprotonates Glu187.

Step 8. Glu187 deprotonates water, which attacks the imine carbon of the covalenlty bound intermediate in a nucleophilic addition.

Step 9. Lys229 deprotonates the formed hydroxyl group, which initiates an elimination of the linear form of D-fructose 1,6-bisphosphate and neutral Lys229.

Step 10. There is evidence in archea that the ring opening/closing reaction is enzyme catalysed by the Lys229. Phosphate deprotonates hydroxyl, and the carbonyl oxygen re-protonates from Lys229, which in turn re-protonates from the phosphate. [PMID:15766250] thus catalysing the ring closure of D-fructose 1,6-bisphosphate.

Products.

Contributors

Gemma L. Holliday, Stuart Lucas, Craig Porter, Gary McDowell, James Willey