Pyruvate, phosphate dikinase

 

Pyruvate phosphate dikinase (PPDK) catalyses the interconversion of ATP, Pi , and pyruvate with AMP, PPi , and PEP. The enzyme has a three-domain structure consisting of consecutive N-terminal, central His455, and C-terminal domains. The nucleotide binding site is located within the N-terminal part of the polypeptide chain, whereas the pyruvate/PEP binding site is located within the C-terminal part of the chain. The first and second partial reactions take place in the active site formed at the interface of the N-terminal domain (which binds Mg(II), ATP, and Ppi) and the central domain (which contributes the catalytic His455) in PPDK conformer 1. The third partial reaction takes place in the active site formed at the interface of the C-terminal domain (which binds Mg(II) and pyruvate) and the central domain (which contributes the phosphorylated His455) in PPDK conformer 2.

 

Reference Protein and Structure

Sequence
P22983 UniProt (2.7.9.1) IPR010121 (Sequence Homologues) (PDB Homologues)
Biological species
[Clostridium] symbiosum (Bacteria) Uniprot
PDB
1kc7 - Pyruvate Phosphate Dikinase with Bound Mg-phosphonopyruvate (2.2 Å) PDBe PDBsum 1kc7
Catalytic CATH Domains
3.30.1490.20 CATHdb 3.20.20.60 CATHdb 3.50.30.10 CATHdb 3.30.470.20 CATHdb (see all for 1kc7)
Cofactors
Water (1), Magnesium(2+) (3) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:2.7.9.1)

ATP(4-)
CHEBI:30616ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
+
pyruvate
CHEBI:15361ChEBI
→
hydron
CHEBI:15378ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
+
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
+
phosphonatoenolpyruvate
CHEBI:58702ChEBI
Alternative enzyme names: PPDK, Orthophosphate dikinase pyruvate, Pyruvate, P(i) dikinase, Pyruvate-inorganic phosphate dikinase, Pyruvate-phosphate dikinase, Pyruvate-phosphate dikinase (phosphorylating), Pyruvate-phosphate ligase, Pyruvic-phosphate dikinase, Pyruvic-phosphate ligase, Pyruvate,phosphate dikinase, Pyruvate,orthophosphate dikinase,

Enzyme Mechanism

Introduction

Phosphoryl transfer is mediated by a carrier histidine, this His455 attacks the beta phosphate of the ATP in a nucleophilic substitution that results in His455 carrying a pyrophosphate moiety and the AMP product. Free phosphate attacks the terminal phosphate of the His455 pyrophosphate moiety in a nucleophilic substitution, resulting in free pyrophosphate and phosphorylated His455. In a proton relay chain involving bulk solvent, Tyr851, water, Ser764 and Cys831 the CH3 group of the pyruvate substrate is deprotonated. This initiates a double bond rearrangement that causes the ketone carbonyl group to attack the phosphorylated His455 in a nucleophilic substitution generating free His455 and the final product.

Catalytic Residues Roles

UniProt PDB* (1kc7)
His455 His455(454)A His455 is the phosphate carrier and initiates nucleophilic substitution on the beta phosphate of ATP leading to His455 carrying a pyrophosphate moiety. In the final step the ketone carbonyl group attacks phosphorylated His455 to generate His455 and phosphoenolpyruvate. covalently attached, nucleofuge, nucleophile, polar interaction
Ser764 Ser764(763)A Receives proton from Cys831 and transports to Tyr851. Proton shuttle. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
Cys831 Cys831(830)A Acts as a general base to deprotonate methyl group of pyruvate substrate- then forms part of proton shuttle. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
Tyr851 Tyr851(850)A End of proton shuttle, transfers proton to solvent. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
Arg337, Arg92, Lys22 Arg337(336)A, Arg92(91)A, Lys22(21)A Electrostatically stabilises the transition state in the formation of pyrophosphorylated His residue. hydrogen bond donor, electrostatic stabiliser
Met103 (main-N), Gly101 (main-N) Met103(102)A (main-N), Gly101(100)A (main-N) Forms oxyanion hole in the initial attack of His455 on beta-phosphate. 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

bimolecular nucleophilic substitution, enzyme-substrate complex formation, intermediate formation, overall reactant used, overall product formed, dephosphorylation, intermediate collapse, assisted keto-enol tautomerisation, proton transfer, proton relay, enzyme-substrate complex cleavage, native state of enzyme regenerated, intermediate terminated

References

  1. Ye D et al. (2001), J Biol Chem, 276, 37630-37639. Investigation of the Catalytic Site within the ATP-Grasp Domain of Clostridium symbiosum Pyruvate Phosphate Dikinase. DOI:10.1074/jbc.m105631200. PMID:11468288.
  2. Herzberg O et al. (2002), Biochemistry, 41, 780-787. Pyruvate site of pyruvate phosphate dikinase: crystal structure of the enzyme-phosphonopyruvate complex, and mutant analysis. DOI:10.1021/bi011799+. PMID:11790099.
  3. McGuire M et al. (1998), Biochemistry, 37, 13463-13474. Location of the Phosphate Binding Site withinClostridium symbiosumPyruvate Phosphate Dikinase†,‡. DOI:10.1021/bi980920i. PMID:9753432.

Step 1. His455 attacks the beta phosphate of the ATP in a nucleophilic substitution that results in His455 carrying a pyrophosphate moiety and the AMP product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg337(336)A electrostatic stabiliser, hydrogen bond donor
His455(454)A polar interaction
Lys22(21)A electrostatic stabiliser, hydrogen bond donor
Gly101(100)A (main-N) electrostatic stabiliser, hydrogen bond donor
Arg92(91)A electrostatic stabiliser, hydrogen bond donor
Met103(102)A (main-N) electrostatic stabiliser, hydrogen bond donor
His455(454)A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, enzyme-substrate complex formation, intermediate formation, overall reactant used, overall product formed

Step 2. Free phosphate attacks the terminal phosphate of the His455 pyrophosphate moiety in a nuclephilic substitution, resulting in free pyrophosphate and phosphorylated His455.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg337(336)A electrostatic stabiliser, hydrogen bond donor
His455(454)A covalently attached
Lys22(21)A electrostatic stabiliser, hydrogen bond donor
Gly101(100)A (main-N) electrostatic stabiliser, hydrogen bond donor
Met103(102)A (main-N) electrostatic stabiliser, hydrogen bond donor

Chemical Components

ingold: bimolecular nucleophilic substitution, dephosphorylation, overall reactant used, intermediate collapse, intermediate formation, overall product formed

Step 3. In a proton relay chain involving bulk solvent, Tyr851, water, Ser764 and Cys831 the CH3 group of the pyruvate substrate is deprotonated. This initiates a double bond rearrangement that causes the ketone carbonyl group to attack the phosphorylated His455 in a nuclephilic substitution generating free His455 and the final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His455(454)A covalently attached
Cys831(830)A proton relay, hydrogen bond acceptor, hydrogen bond donor
Ser764(763)A proton relay, hydrogen bond acceptor, hydrogen bond donor
Tyr851(850)A proton relay, hydrogen bond acceptor, hydrogen bond donor
Tyr851(850)A proton acceptor
Ser764(763)A proton donor, proton acceptor
Cys831(830)A proton acceptor
Tyr851(850)A proton donor
His455(454)A nucleofuge
Cys831(830)A proton donor

Chemical Components

ingold: bimolecular nucleophilic substitution, assisted keto-enol tautomerisation, proton transfer, proton relay, enzyme-substrate complex cleavage, native state of enzyme regenerated, intermediate collapse, intermediate terminated, overall reactant used, overall product formed
Download: Image, Marvin File

Step 1. His455 attacks the beta phosphate of the ATP in a nucleophilic substitution that results in His455 carrying a pyrophosphate moiety and the AMP product.

Step 2. Free phosphate attacks the terminal phosphate of the His455 pyrophosphate moiety in a nuclephilic substitution, resulting in free pyrophosphate and phosphorylated His455.

Step 3. In a proton relay chain involving bulk solvent, Tyr851, water, Ser764 and Cys831 the CH3 group of the pyruvate substrate is deprotonated. This initiates a double bond rearrangement that causes the ketone carbonyl group to attack the phosphorylated His455 in a nuclephilic substitution generating free His455 and the final product.

Products.

Contributors

André Minoche, Gemma L. Holliday, Anna Waters, Craig Porter