Pyruvate carboxylase

 

Pyruvate carboxylase, isolated from Rhizobium etli, is a biotin-dependent enzyme. It catalyses the carboxylation of pyruvate to oxaloacetate using bicarbonate and coupled to the hydrolysis of ATP to ADP and phosphate. This reaction occurs in two major steps: the carboxylation of biotin by ATP and bicarbonate in the biotin carboxylase (BC) domain, and the carboxylation of pyruvate in the carboxyltransferase (CT) domain. The biotin is bound to the biotin carboxyl carrier protein (BCCP) domain, which moves between the BC domain of one subunit and the CT domain of a neighbouring subunit.

 

Reference Protein and Structure

Sequence
Q2K340 UniProt (6.4.1.1) IPR005930 (Sequence Homologues) (PDB Homologues)
Biological species
Rhizobium etli CFN 42 (Bacteria) Uniprot
PDB
2qf7 - Crystal structure of a complete multifunctional pyruvate carboxylase from Rhizobium etli (2.0 Å) PDBe PDBsum 2qf7
Catalytic CATH Domains
3.20.20.70 CATHdb 1.10.472.90 CATHdb 3.30.470.20 CATHdb (see all for 2qf7)
Cofactors
Biotinate (1), Zinc(2+) (1), Magnesium(2+) (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.4.1.1)

hydrogencarbonate
CHEBI:17544ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
+
pyruvate
CHEBI:15361ChEBI
→
hydron
CHEBI:15378ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
+
oxaloacetate(2-)
CHEBI:16452ChEBI
Alternative enzyme names: Pyruvic carboxylase,

Enzyme Mechanism

Introduction

BC domain: The bicarbonate is deprotonated by Glu305 and then acts as the nucleophile for attack on ATP to form ADP and phosphorylated bicarbonate. The latter undergoes spontaneous decarboxylation to form carbon dioxide and phosphate. The phosphate removes a proton from biotin to produce the biotin amidate. The carbonyl of biotin reforms and the activated biotin acts as a nucleophile through its nitrogen to attack the carbon dioxide to form carboxylbiotin.

CT domain: Pyruvate binds to the zinc ion through the oxygen of the carbonyl group. Carbon dioxide is then liberated from the biotin moiety. The activates biotin then abstracts a proton from pyruvate via Thr882. The activate substrate then initiates a nucleophilic attack on the carbon dioxide to form the final product.

Catalytic Residues Roles

UniProt PDB* (2qf7)
His747, His749, Asp549, Asp655 His747(758)A, His749(760)A, Asp549(560)A, Asp655(666)A Coordinates to the zinc ion, activating it to stabilise the formation of the pyruvate enolate. metal ligand
Glu305 Glu305(316)A Acts as a general acid/base in the BC domain reaction. proton acceptor, electrostatic stabiliser, proton donor
Arg353 Arg353(364)A Helps stabilise and activate the Glu305 as a general acid/base. activator, electrostatic stabiliser
Thr882 Thr882(893)A Acts as a general acid/base in a proton relay between biotin and pyruvate in the CT domain. proton relay, proton acceptor, proton donor
Glu297 Glu297(308)A Forms part of the binding site for both magnesium ions in the BC domain. metal ligand
Glu283 Glu283(294)A Forms part of the binding site for magnesium 1 in the BC domain metal ligand
Asn299 Asn299(310)A Forms part of the binding site for magnesium 2 in the BC domain. metal ligand
Lys718 Lys718(729)A Helps stabilise the biotin intermediates. hydrogen bond donor, 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

proton transfer, bimolecular nucleophilic substitution, overall reactant used, intermediate formation, overall product formed, unimolecular elimination by the conjugate base, intermediate collapse, decarboxylation, assisted tautomerisation (not keto-enol), cofactor used, intermediate terminated, bimolecular nucleophilic addition, native state of enzyme is not regenerated, assisted keto-enol tautomerisation, native state of cofactor regenerated, inferred reaction step, native state of enzyme regenerated

References

  1. Menefee AL et al. (2014), FEBS J, 281, 1333-1354. Nearly 50 years in the making: defining the catalytic mechanism of the multifunctional enzyme, pyruvate carboxylase. DOI:10.1111/febs.12713. PMID:24476417.
  2. Lietzan AD et al. (2014), Arch Biochem Biophys, 562, 70-79. The role of biotin and oxamate in the carboxyltransferase reaction of pyruvate carboxylase. DOI:10.1016/j.abb.2014.08.008. PMID:25157442.
  3. Adina-Zada A et al. (2014), Biochemistry, 53, 1051-1058. Coordinating role of His216 in MgATP binding and cleavage in pyruvate carboxylase. DOI:10.1021/bi4016814. PMID:24460480.
  4. Lietzan AD et al. (2013), Biochem Biophys Res Commun, 441, 377-382. Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs. DOI:10.1016/j.bbrc.2013.10.066. PMID:24157795.
  5. Lasso G et al. (2010), Structure, 18, 1300-1310. Cryo-EM analysis reveals new insights into the mechanism of action of pyruvate carboxylase. DOI:10.1016/j.str.2010.07.008. PMID:20947019.
  6. Duangpan S et al. (2010), Biochemistry, 49, 3296-3304. Probing the catalytic roles of Arg548 and Gln552 in the carboxyl transferase domain of the Rhizobium etli pyruvate carboxylase by site-directed mutagenesis. DOI:10.1021/bi901894t. PMID:20230056.
  7. Jitrapakdee S et al. (2008), Biochem J, 413, 369-387. Structure, mechanism and regulation of pyruvate carboxylase. DOI:10.1042/bj20080709. PMID:18613815.

Step 1. Reaction occurs in the BC domain. The bicarbonate is deprotonated by Glu305 and then acts as a nucleophile and attacks the gamma-phosphate in a substitution reaction, liberating ADP. Mg(II) stabilises/activates the ATP

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg353(364)A activator
Arg353(364)A electrostatic stabiliser
Glu297(308)A metal ligand
Asn299(310)A metal ligand
Glu283(294)A metal ligand
Glu305(316)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, intermediate formation, overall product formed

Step 2. Reaction occurs in the BC domain. The phosphorylated bicarbonate undergoes a decarboxylation reaction (E1cb) to liberate carbon dioxide and phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg353(364)A electrostatic stabiliser
Glu305(316)A electrostatic stabiliser
Glu297(308)A metal ligand
Asn299(310)A metal ligand
Glu283(294)A metal ligand

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse, intermediate formation, decarboxylation

Step 3. Reaction occurs in the BC domain. The phosphate deprotonates one of the N-H groups of biotin with concomitant tautomerisation to produce an oxyanion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu297(308)A metal ligand
Asn299(310)A metal ligand
Glu283(294)A metal ligand

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol), cofactor used, intermediate formation, intermediate terminated, overall product formed

Step 4. Reaction occurs in the BC domain. The oxyanion re-forms the carbonyl group, causing the C=N bond of the activated biotin to add to the carbon dioxide in a nucleophilic manner.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg353(364)A electrostatic stabiliser
Glu305(316)A electrostatic stabiliser
Glu297(308)A metal ligand
Asn299(310)A metal ligand
Glu283(294)A metal ligand

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation, native state of enzyme is not regenerated

Step 5. Reaction occurs in the CT domain. Carbon dioxide is eliminated from the biotin moiety.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys718(729)A hydrogen bond donor, electrostatic stabiliser
Asp549(560)A metal ligand
Asp655(666)A metal ligand
His749(760)A metal ligand
His747(758)A metal ligand

Chemical Components

intermediate formation, ingold: unimolecular elimination by the conjugate base, decarboxylation

Step 6. The activated biotin moiety abstracts a proton from Thr882, which in turn abstracts a proton from the pyruvate substrate forming the enol intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys718(729)A electrostatic stabiliser
Asp549(560)A metal ligand
Asp655(666)A metal ligand
His749(760)A metal ligand
His747(758)A metal ligand
Thr882(893)A proton donor, proton acceptor, proton relay

Chemical Components

assisted keto-enol tautomerisation, proton transfer, native state of cofactor regenerated, overall reactant used

Step 7. The enolate intermediate initiates a nucleophilic attack on the carbon dioxide molecule to form the final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp549(560)A metal ligand
Asp655(666)A metal ligand
His749(760)A metal ligand
His747(758)A metal ligand

Chemical Components

ingold: bimolecular nucleophilic addition, overall product formed

Step 8. Inferred return step to regenerate the active site of the BC domain.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg353(364)A electrostatic stabiliser
Glu297(308)A metal ligand
Glu283(294)A metal ligand
Asn299(310)A metal ligand
Glu305(316)A proton donor

Chemical Components

proton transfer, inferred reaction step, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Reaction occurs in the BC domain. The bicarbonate is deprotonated by Glu305 and then acts as a nucleophile and attacks the gamma-phosphate in a substitution reaction, liberating ADP. Mg(II) stabilises/activates the ATP

Step 2. Reaction occurs in the BC domain. The phosphorylated bicarbonate undergoes a decarboxylation reaction (E1cb) to liberate carbon dioxide and phosphate.

Step 3. Reaction occurs in the BC domain. The phosphate deprotonates one of the N-H groups of biotin with concomitant tautomerisation to produce an oxyanion.

Step 4. Reaction occurs in the BC domain. The oxyanion re-forms the carbonyl group, causing the C=N bond of the activated biotin to add to the carbon dioxide in a nucleophilic manner.

Step 5. Reaction occurs in the CT domain. Carbon dioxide is eliminated from the biotin moiety.

Step 6. The activated biotin moiety abstracts a proton from Thr882, which in turn abstracts a proton from the pyruvate substrate forming the enol intermediate.

Step 7. The enolate intermediate initiates a nucleophilic attack on the carbon dioxide molecule to form the final product.

Step 8. Inferred return step to regenerate the active site of the BC domain.

Products.

Introduction

BC domain: The bicarbonate is deprotonated by a water molecule and then acts as the nucleophile for attack on ATP to form ADP and phosphorylated bicarbonate. The latter undergoes spontaneous decarboxylation to form carbon dioxide and phosphate. The phosphate removes a proton from biotin to produce the biotin amidate. The carbonyl of biotin reforms and the activated biotin acts as a nucleophile through its nitrogen to attack the carbon dioxide to form carboxylbiotin.

CT domain: Pyruvate binds to the zinc ion through the oxygen of the carbonyl group. Asp549 removes a proton from pyruvate to form the enolate. The enolate then attacks the carboxylate of carboxylbiotin in a nucleophilic substitution. This produces oxaloacetate and the biotin amidate, which is stabilised by accepting a proton from Lys718 to produce the isourea form of biotin. Lys718 then removes this proton and biotin returns to the ureido form by accepting a proton from Asp549.

Catalytic Residues Roles

UniProt PDB* (2qf7)
Asp549 Asp549(560)A Asp549 acts as a base to produce the enolate form of pyruvate. Later it acts as an acid to return biotin to its ureido form. hydrogen bond acceptor, hydrogen bond donor, metal ligand, proton acceptor, proton donor
His747, His749, Asp655 His747(758)A, His749(760)A, Asp655(666)A Coordinates to the zinc ion, activating it to stabilise the formation of the pyruvate enolate. metal ligand
Glu305, Arg353, Thr882 Glu305(316)A, Arg353(364)A, Thr882(893)A These residues have no annotated function in this mechanism proposal.
Glu297 Glu297(308)A Forms part of the binding site for both magnesium ions in the BC domain. metal ligand
Glu283 Glu283(294)A Forms part of the binding site for magnesium 1 in the BC domain metal ligand
Asn299 Asn299(310)A Forms part of the binding site for magnesium 2 in the BC domain. metal ligand
Lys718 Lys718(729)A Lys718 acts as an acid during the formation of the enol form of biotin in the PC reaction. It then acts as a base and removes this proton so that the enolate form of biotin can act as a nucleophile. hydrogen bond acceptor, hydrogen bond donor, 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 substitution, overall reactant used, intermediate formation, overall product formed, unimolecular elimination by the conjugate base, intermediate collapse, decarboxylation, assisted tautomerisation (not keto-enol), cofactor used, intermediate terminated, bimolecular nucleophilic addition, native state of enzyme is not regenerated, assisted keto-enol tautomerisation, native state of cofactor regenerated, native state of enzyme regenerated

References

  1. Jitrapakdee S et al. (2008), Biochem J, 413, 369-387. Structure, mechanism and regulation of pyruvate carboxylase. DOI:10.1042/bj20080709. PMID:18613815.
  2. Menefee AL et al. (2014), FEBS J, 281, 1333-1354. Nearly 50 years in the making: defining the catalytic mechanism of the multifunctional enzyme, pyruvate carboxylase. DOI:10.1111/febs.12713. PMID:24476417.
  3. Zeczycki TN et al. (2009), Biochemistry, 48, 4305-4313. Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli. DOI:10.1021/bi9003759. PMID:19341298.
  4. Adina-Zada A et al. (2008), Int J Biochem Cell Biol, 40, 1743-1752. Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme. DOI:10.1016/j.biocel.2008.01.001. PMID:18272421.
  5. St Maurice M et al. (2007), Science, 317, 1076-1079. Domain Architecture of Pyruvate Carboxylase, a Biotin-Dependent Multifunctional Enzyme. DOI:10.1126/science.1144504. PMID:17717183.
  6. Yong-Biao J et al. (2004), Biochemistry, 43, 5912-5920. Identification of the Catalytic Residues Involved in the Carboxyl Transfer of Pyruvate Carboxylase. DOI:10.1021/bi035783q. PMID:15134465.

Step 1. Reaction occurs in the BC domain. The bicarbonate is deprotonated by a hydroxide/water molecule and then acts as a nucleophile and attacks the gamma-phosphate in a substitution reaction, liberating ADP. Mg(II) stabilises/activates the ATP

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu297(308)A metal ligand
Glu283(294)A metal ligand
Asn299(310)A metal ligand

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, intermediate formation, overall product formed

Step 2. Reaction occurs in the BC domain. The phosphorylated bicarbonate undergoes a decarboxylation reaction (E1cb) to liberate carbon dioxide and phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu297(308)A metal ligand
Glu283(294)A metal ligand
Asn299(310)A metal ligand

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse, intermediate formation, decarboxylation

Step 3. Reaction occurs in the BC domain. The phosphate deprotonates one of the N-H groups of biotin with concomitant tautomerisation to produce an oxyanion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu297(308)A metal ligand
Glu283(294)A metal ligand
Asn299(310)A metal ligand

Chemical Components

proton transfer, assisted tautomerisation (not keto-enol), cofactor used, intermediate formation, intermediate terminated, overall product formed

Step 4. Reaction occurs in the BC domain. The oxyanion re-forms the carbonyl group, causing the C=N bond of the activated biotin to add to the carbon dioxide in a nucleophilic manner.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu297(308)A metal ligand
Glu283(294)A metal ligand
Asn299(310)A metal ligand

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation, native state of enzyme is not regenerated

Step 5. Reaction occurs in the CT domain. The Asp549 deprotonates pyruvate, activating the pyruvate in a keto-enol tautomerisation.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys718(729)A hydrogen bond donor
Asp549(560)A hydrogen bond acceptor
Asp655(666)A activator
Asp549(560)A metal ligand
Asp655(666)A metal ligand
His749(760)A metal ligand
His747(758)A metal ligand
Asp549(560)A proton acceptor

Chemical Components

proton transfer, assisted keto-enol tautomerisation, intermediate formation, overall reactant used

Step 6. Reaction occurs in the CT domain. The activated pyruvate attacks the carboxylated biotin in a nucleophilic substitution, producing the oxaloacetate product and activated biotin.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys718(729)A hydrogen bond donor
Asp655(666)A activator
Asp549(560)A metal ligand
Asp655(666)A metal ligand
His749(760)A metal ligand
His747(758)A metal ligand
Lys718(729)A proton donor

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, overall product formed, intermediate collapse, intermediate terminated

Step 7. Reaction occurs in the CT domain. Lys718 deprotonates the activated biotin, which in turn deprotonates Asp549, returning the enzyme to its ground state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys718(729)A hydrogen bond acceptor
Asp549(560)A hydrogen bond donor
Asp549(560)A metal ligand
Asp655(666)A metal ligand
His749(760)A metal ligand
His747(758)A metal ligand
Lys718(729)A proton acceptor
Asp549(560)A proton donor

Chemical Components

proton transfer, native state of cofactor regenerated, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Reaction occurs in the BC domain. The bicarbonate is deprotonated by a hydroxide/water molecule and then acts as a nucleophile and attacks the gamma-phosphate in a substitution reaction, liberating ADP. Mg(II) stabilises/activates the ATP

Step 2. Reaction occurs in the BC domain. The phosphorylated bicarbonate undergoes a decarboxylation reaction (E1cb) to liberate carbon dioxide and phosphate.

Step 3. Reaction occurs in the BC domain. The phosphate deprotonates one of the N-H groups of biotin with concomitant tautomerisation to produce an oxyanion.

Step 4. Reaction occurs in the BC domain. The oxyanion re-forms the carbonyl group, causing the C=N bond of the activated biotin to add to the carbon dioxide in a nucleophilic manner.

Step 5. Reaction occurs in the CT domain. The Asp549 deprotonates pyruvate, activating the pyruvate in a keto-enol tautomerisation.

Step 6. Reaction occurs in the CT domain. The activated pyruvate attacks the carboxylated biotin in a nucleophilic substitution, producing the oxaloacetate product and activated biotin.

Step 7. Reaction occurs in the CT domain. Lys718 deprotonates the activated biotin, which in turn deprotonates Asp549, returning the enzyme to its ground state.

Products.

Contributors

Julia D. Fischer, Gemma L. Holliday