Transketolase

 

Transketolase (EC:2.2.1.1) (TK) catalyses the reversible transfer of a two-carbon ketol unit from xylulose 5-phosphate to an aldose receptor, such as ribose 5-phosphate, to form sedoheptulose 7-phosphate and glyceraldehyde 3- phosphate. This enzyme, together with transaldolase, provides a link between the glycolytic and pentose-phosphate pathways. TK requires thiamine pyrophosphate and a divalent metal cation as a cofactor.

Catalytic activity has been noted with a wide variety of divalent metal ions coordinating the diphosphate group of the cofactor [PMID:9924800] and whilst there is still some debate as to the exact nature of the native metal cation (most of the work on the determination of the mechanism has utilised the magnesium cation), Esakova et al. have determined that the enzyme is more stable in the presence of Ca(II), making this the more likely native metal cation [PMID:16125202] .

 

Reference Protein and Structure

Sequence
P29401 UniProt (2.2.1.1) IPR005478 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
4kxv - Human transketolase in covalent complex with donor ketose D-xylulose-5-phosphate, crystal 1 (0.97 Å) PDBe PDBsum 4kxv
Catalytic CATH Domains
3.40.50.970 CATHdb (see all for 4kxv)
Cofactors
Magnesium(2+) (1), Thiamine(1+) diphosphate(3-) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:2.2.1.1)

D-glyceraldehyde 3-phosphate(2-)
CHEBI:59776ChEBI
+
sedoheptulose 7-phosphate(2-)
CHEBI:57483ChEBI
→
D-xylulose 5-phosphate(2-)
CHEBI:57737ChEBI
+
aldehydo-D-ribose 5-phosphate(2-)
CHEBI:58273ChEBI
Alternative enzyme names: Glycolaldehydetransferase, Glycoaldehyde transferase,

Enzyme Mechanism

Introduction

Glu418B activates the thiamine diphosphate cofactor by abstracting a proton from the NH group of the 6-membered ring. This results in double bond rearrangement and the abstraction of a proton from the N=CH-S moiety. The carbanion of thiamine diphosphate then attacks the carbonyl carbon of the sugar (in its open form) in a nucleophilic addition that results in the cofactor undergoing another double bond rearrangement and abstracting the proton back from Glu418B. His263A deprotonates the 2-OH group of the covaltnely attached intermediate, initiating the elimination of D-xylulose 5-phosphate. Thaimine diphosphate acts as an electron sink. Thiamine diphosphate initiates a double bond rearrangement, which results in the intermediate attacking (2R)-2-Hydroxy-3-(phosphonooxy)-propanal in a nucleophilic addition. The formed oxyanion deprotonates His263A. Glu418B deprotonates thiamine diphosphate, which initiates a double bond rearrangement, that deprotonates the hydroxide of the intermediate, and results in a reformation of the carbanionic activated cofactor and the D-xylulose 5-phosphate product. The carbanion of the thiamine diphosphate cofactor deprotonates the adjacent amine, which initiates double bond rearrangement that results in the deprotonation of Glu418B. Cyclisation of the sugar product occurs spontaneously outside of the enzyme's active site.

Catalytic Residues Roles

UniProt PDB* (4kxv)
Glu418 Glu418B Acts as a general acid/base, important for activating the thiamine diphosphate cofactor. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
His263 His263A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
His30, His481 His30A, His481B Help bind and stabilise the substrate and reactive intermediates formed during the course of the reaction. activator, hydrogen bond donor, steric role
*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, assisted tautomerisation (not keto-enol), cofactor used, bimolecular nucleophilic addition, aldol addition, overall reactant used, intermediate formation, bimolecular elimination, overall product formed, intermediate collapse, intermediate terminated, native state of cofactor regenerated, native state of enzyme regenerated, reaction occurs outside the enzyme, cyclisation, intramolecular nucleophilic addition

References

  1. Wikner C et al. (1997), Biochemistry, 36, 15643-15649. Identification of Catalytically Important Residues in Yeast Transketolase†. DOI:10.1021/bi971606b. PMID:9398292.
  2. Nauton L et al. (2016), Biochemistry, 55, 2144-2152. Insights into the Thiamine Diphosphate Enzyme Activation Mechanism: Computational Model for Transketolase Using a Quantum Mechanical/Molecular Mechanical Method. DOI:10.1021/acs.biochem.5b00787. PMID:26998737.
  3. Tittmann K (2014), Bioorg Chem, 57, 263-280. Sweet siblings with different faces: the mechanisms of FBP and F6P aldolase, transaldolase, transketolase and phosphoketolase revisited in light of recent structural data. DOI:10.1016/j.bioorg.2014.09.001. PMID:25267444.
  4. Sheng X et al. (2013), J Mol Graph Model, 39, 23-28. Theoretical studies on the common catalytic mechanism of transketolase by using simplified models. DOI:10.1016/j.jmgm.2012.11.001. PMID:23220278.
  5. Esakova OA et al. (2005), Life Sci, 78, 8-13. Effects of transketolase cofactors on its conformation and stability. DOI:10.1016/j.lfs.2004.12.055. PMID:16125202.
  6. Schenk G et al. (1998), Int J Biochem Cell Biol, 30, 1297-1318. Properties and functions of the thiamin diphosphate dependent enzyme transketolase. DOI:10.1016/s1357-2725(98)00095-8. PMID:9924800.

Step 1. Glu418B deprotonates the thiamine diphosphate cofactor, which initiates double bond rearrangement that results in the deprotonation of the N=CH-S group, activating the cofactor. The double bonds can rearrage either as described here, or anti-clockwise around the aromatic ring.

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Catalytic Residues Roles

Residue Roles
His481B electrostatic stabiliser, hydrogen bond acceptor
His263A hydrogen bond donor
Glu418B hydrogen bond acceptor
Glu418B proton acceptor

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol), cofactor used

Step 2. The carbanion of thiamine diphosphate attacks the carbonyl carbon of sedoheptulose 7-phosphate in a nucleophilic addition that results in the cofactor undergoing double bond rearrangement that results in the deprotonation of Glu418B.

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Catalytic Residues Roles

Residue Roles
Glu418B hydrogen bond donor
His481B electrostatic stabiliser, hydrogen bond acceptor
His30A hydrogen bond donor
His263A hydrogen bond donor
Glu418B proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, aldol addition, overall reactant used, intermediate formation

Step 3. His263A deprotonates the 2-OH group of the covalently attached intermediate, initiating the elimination of D-xylulose 5-phosphate. Thaimine diphosphate acts as an electron sink. His30 facilitates the proton transfer to His263 by a hydrogen bond to the hydroxyl group, positioning it in the correct orientation and stabilising the developing negative charge of the transition state [PMID:9398292]. The cofactor acts as an electron sink.

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Catalytic Residues Roles

Residue Roles
Glu418B hydrogen bond acceptor
His481B electrostatic stabiliser, hydrogen bond acceptor
His30A hydrogen bond donor, activator, steric role
His263A hydrogen bond donor, hydrogen bond acceptor
His263A proton acceptor

Chemical Components

ingold: bimolecular elimination, assisted tautomerisation (not keto-enol), overall product formed, intermediate formation, intermediate collapse

Step 4. Thiamine diphosphate initiates a double bond rearrangement, which results in the intermediate attacking (2R)-2-Hydroxy-3-(phosphonooxy)-propanal in a nucleophilic addition. The formed oxyanion deprotonates His263A.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu418B hydrogen bond acceptor
His481B electrostatic stabiliser, hydrogen bond acceptor
His30A hydrogen bond donor, activator, steric role
His263A hydrogen bond donor
His263A proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, assisted tautomerisation (not keto-enol), aldol addition, overall reactant used, intermediate formation

Step 5. Glu418B deprotonates thiamine diphosphate, which initiates a double bond rearrangement, that deprotonates the hydroxide of the intermediate, and results in a reformation of the carbanionic activated cofactor and the D-xylulose-5-phosphate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu418B hydrogen bond acceptor
His481B electrostatic stabiliser, hydrogen bond acceptor
His30A hydrogen bond donor
His263A hydrogen bond donor
Glu418B proton acceptor

Chemical Components

proton transfer, ingold: bimolecular elimination, assisted tautomerisation (not keto-enol), overall product formed, intermediate collapse, intermediate formation

Step 6. The carbanion of the thiamine diphosphate cofactor deprotonates the adjacent amine, which initiates double bond rearrangement that results in the deprotonation of Glu418B.

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Catalytic Residues Roles

Residue Roles
Glu418B hydrogen bond donor
His481B electrostatic stabiliser, hydrogen bond acceptor
His30A hydrogen bond donor
His263A hydrogen bond donor
Glu418B proton donor

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol), intermediate terminated, native state of cofactor regenerated, native state of enzyme regenerated

Step 7. The product of the enzyme is the linear molecule. The cyclisation is not enzyme catalysed, and has been inferred from the overall reaction products.

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Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, cyclisation, ingold: intramolecular nucleophilic addition, proton transfer
Download: Image, Marvin File

Step 1. Glu418B deprotonates the thiamine diphosphate cofactor, which initiates double bond rearrangement that results in the deprotonation of the N=CH-S group, activating the cofactor. The double bonds can rearrage either as described here, or anti-clockwise around the aromatic ring.

Step 2. The carbanion of thiamine diphosphate attacks the carbonyl carbon of sedoheptulose 7-phosphate in a nucleophilic addition that results in the cofactor undergoing double bond rearrangement that results in the deprotonation of Glu418B.

Step 3. His263A deprotonates the 2-OH group of the covalently attached intermediate, initiating the elimination of D-xylulose 5-phosphate. Thaimine diphosphate acts as an electron sink. His30 facilitates the proton transfer to His263 by a hydrogen bond to the hydroxyl group, positioning it in the correct orientation and stabilising the developing negative charge of the transition state [PMID:9398292]. The cofactor acts as an electron sink.

Step 4. Thiamine diphosphate initiates a double bond rearrangement, which results in the intermediate attacking (2R)-2-Hydroxy-3-(phosphonooxy)-propanal in a nucleophilic addition. The formed oxyanion deprotonates His263A.

Step 5. Glu418B deprotonates thiamine diphosphate, which initiates a double bond rearrangement, that deprotonates the hydroxide of the intermediate, and results in a reformation of the carbanionic activated cofactor and the D-xylulose-5-phosphate product.

Step 6. The carbanion of the thiamine diphosphate cofactor deprotonates the adjacent amine, which initiates double bond rearrangement that results in the deprotonation of Glu418B.

Step 7. The product of the enzyme is the linear molecule. The cyclisation is not enzyme catalysed, and has been inferred from the overall reaction products.

Products.

Introduction

Activated carbanion of thiamine diphosphate (ThDP) cofactor performs a nucleophilic attack towards the D-xylulose-5-phosphate, resulting in double bond rearrangement concerted with deprotonation of the ThDP's amine group by the substrate. Lys244 deprotonates His258 which then deprotonates the 2-OH group of the covalently attached intermediate, initiating the elimination of D-glyceraldehyde 3-phosphate. Thiamine diphosphate acts as an electron sink. Thiamine diphosphate initiates a double bond rearrangement, which results in the intermediate attacking the aldose D-erythrose-4-phosphate substrate in a nucleophilic addition. This results in a more negative carbonyl oxygen which is subsequently implicated in a series of proton transfers for restoration of the initial protonation state of His258 and Lys244 catalytic residues. ThDP cofactor initiates a double bond rearrangement that deprotonates the hydroxide of the intermediate, and results in a reformation of the carbanionic activated cofactor and the D-fructose-6-phosphate product.

Catalytic Residues Roles

UniProt PDB* (4kxv)
His258 His258A His258 operates as the general base and acid to mediate proton transfer by accepting a proton from the O3H group of the substrate and delivering another one to Lys244. proton acceptor, proton donor
Lys244 Lys244A Involve in acid-base catalysis by accepting and donating protons from His258. 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

bimolecular nucleophilic addition, proton transfer, bimolecular elimination, assisted keto-enol tautomerisation, intermediate collapse, intermediate formation, overall product formed, overall reactant used, assisted tautomerisation (not keto-enol), aldol addition

References

  1. Prejanò M et al. (2019), Chemphyschem, 20, 2881-2886. The Catalytic Mechanism of Human Transketolase. DOI:10.1002/cphc.201900650. PMID:31489766.
  2. Prejanò M et al. (2020), ACS Catal, 10, 2872-2881. How the Destabilization of a Reaction Intermediate Affects Enzymatic Efficiency: The Case of Human Transketolase. DOI:10.1021/acscatal.9b04690. PMID:33828899.

Step 1. The carbanion of thiamine diphosphate (ThDP) attacks the carbonyl carbon of D-xylulose-5-phosphate in a nucleophilic addition that results in the cofactor undergoing double bond rearrangement. Simultaneously, the carbonyl oxygen of D-xylulose-5-phosphate deprotonates the amine group of the ThDP cofactor.

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Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 2. Lys244 deprotonates His258, thereby activating it. Activated His258 deprotonates the 2-OH group of the covalently attached intermediate, initiating the elimination of D-glyceraldehyde 3-phosphate via cleavage of the C-C scissile bond. Thiamine diphosphate acts as an electron sink.

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Catalytic Residues Roles

Residue Roles
Lys244A proton acceptor
His258A proton donor, proton acceptor

Chemical Components

ingold: bimolecular elimination, assisted keto-enol tautomerisation, intermediate collapse, intermediate formation, overall product formed

Step 3. Formation of D-glyceraldehyde-3-phosphate product.

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Catalytic Residues Roles

Residue Roles

Chemical Components

overall product formed

Step 4. Thiamine diphosphate initiates a double bond rearrangement, which results in the intermediate attacking the aldose D-erythrose-4-phosphate substrate in a nucleophilic addition. This resulted in a more negative carbonyl oxygen which is subsequently implicated in a series of proton transfers for restoration of the initial protonation state of His258 and Lys244 catalytic residues.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys244A proton donor
His258A proton donor, proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation, assisted tautomerisation (not keto-enol), aldol addition

Step 5. ThDP cofactor initiates a double bond rearrangement, that deprotonates the hydroxide of the intermediate, and results in a reformation of the carbanionic activated cofactor and the D-fructose-6-phosphate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

intermediate collapse, overall product formed, ingold: bimolecular elimination, proton transfer
Download: Image, Marvin File

Step 1. The carbanion of thiamine diphosphate (ThDP) attacks the carbonyl carbon of D-xylulose-5-phosphate in a nucleophilic addition that results in the cofactor undergoing double bond rearrangement. Simultaneously, the carbonyl oxygen of D-xylulose-5-phosphate deprotonates the amine group of the ThDP cofactor.

Step 2. Lys244 deprotonates His258, thereby activating it. Activated His258 deprotonates the 2-OH group of the covalently attached intermediate, initiating the elimination of D-glyceraldehyde 3-phosphate via cleavage of the C-C scissile bond. Thiamine diphosphate acts as an electron sink.

Step 3. Formation of D-glyceraldehyde-3-phosphate product.

Step 4. Thiamine diphosphate initiates a double bond rearrangement, which results in the intermediate attacking the aldose D-erythrose-4-phosphate substrate in a nucleophilic addition. This resulted in a more negative carbonyl oxygen which is subsequently implicated in a series of proton transfers for restoration of the initial protonation state of His258 and Lys244 catalytic residues.

Step 5. ThDP cofactor initiates a double bond rearrangement, that deprotonates the hydroxide of the intermediate, and results in a reformation of the carbanionic activated cofactor and the D-fructose-6-phosphate product.

Products.

Contributors

Christian Drew, Craig Porter, Charity Hornby, Trung Nguyen, Gemma L. Holliday