Nucleoside-diphosphate kinase

 

Nucleoside diphosphate kinase (NDP kinase) is responsible for phosphorylating non-adenine nucleoside diphosphates. The phosphate donor is usually ATP, but the diphosphate nucleotide substrate is non-specific for either base or ribose / deoxyribose.

The mechanism of nucleotide binding is different from that of most other phoshokinases and nucleotide binding proteins which use a parallel beta-sheet, NDP using an anti-parallel beta-sheet. NDPK from Dictyostelium discoideum is used as a model because it is particularly easy to crystallise.

 

Reference Protein and Structure

Sequence
P22887 UniProt (2.7.4.6) IPR001564 (Sequence Homologues) (PDB Homologues)
Biological species
Dictyostelium discoideum (Slime mould) Uniprot
PDB
1ndp - ADENOSINE 5'-DIPHOSPHATE BINDING AND THE ACTIVE SITE OF NUCLEOSIDE DIPHOSPHATE KINASE (2.2 Å) PDBe PDBsum 1ndp
Catalytic CATH Domains
3.30.70.141 CATHdb (see all for 1ndp)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:2.7.4.6)

ATP(4-)
CHEBI:30616ChEBI
+
nucleoside 5'-diphosphate(3-)
CHEBI:57930ChEBI
→
nucleoside triphosphate(4-)
CHEBI:61557ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
Alternative enzyme names: UDP kinase, Nucleoside 5'-diphosphate kinase, Nucleoside diphosphate (UDP) kinase, Nucleoside diphosphokinase, Nucleotide phosphate kinase, Uridine diphosphate kinase, Nucleoside 5'-diphosphate phosphotransferase, NDK,

Enzyme Mechanism

Introduction

NDPK operates using a 'ping-pong' mechanism. The gamma-phosphate of ATP is transferred to the delta-N atom of the doubly protonated His 122 of NDPK, with the delta-N proton concomitantly transferred to the gamma-phosphate. A nucleoside diphosphate binds in the active site. The phosphate group is transferred from His 122-P to the 5' hydroxyl of the ribose. Both phosphoryl transfers are dissociative SN2-like reactions, similar to the uncatalysed reaction.

The 3' ribose / deoxyribose hydroxyl group is more catalytically important than Tyr 56 or Lys 16; in the first transfer to His 122, it hydrogen bonds to the gamma-phosphate group, forcing the substrate into a reactive conformation and stabilising the negative charge by hydrogen bonding. The enzymatic reaction can therefore be described as substrate-assisted.

Catalytic Residues Roles

UniProt PDB* (1ndp)
Asn119 Asn119B Ensures that the substrates are bound in the correct position. hydrogen bond acceptor, hydrogen bond donor, steric role, electrostatic stabiliser
His122 His122B Acts as a general acid/base and the catalytic nucleophile. It is phosphrylated during the course of the reaction. hydrogen bond donor, nucleophile, proton acceptor, proton donor, nucleofuge
Glu133 Glu133B Activates the catalytic histidine as part of a Glu-His dyad. increase nucleophilicity, hydrogen bond acceptor, steric role
Lys16 Lys16B Lys 16 stabilises the negative charge on the phosphoryl group, lowering the transition state energy. Lys 16 also plays a role in keeping Tyr 56 protonated. hydrogen bond donor, electrostatic stabiliser
Tyr56 Tyr56B Tyr 56 stabilises the phosphohistidine intermediate by hydrogen bonding to phosphohistidine. 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, enzyme-substrate complex formation, overall product formed, intermediate formation, enzyme-substrate complex cleavage, intermediate collapse, intermediate terminated, native state of enzyme regenerated

References

  1. Schneider B et al. (2001), Eur J Biochem, 268, 1964-1971. Mechanism of phosphoryl transfer by nucleoside diphosphate kinase pH dependence and role of the active site Lys16 and Tyr56 residues. PMID:11277918.
  2. Pédelacq JD et al. (2005), Protein Sci, 14, 2562-2573. Structural and functional features of an NDP kinase from the hyperthermophile crenarchaeon Pyrobaculum aerophilum. DOI:10.1110/ps.051664205. PMID:16195547.
  3. Gallois-Montbrun S et al. (2003), Mol Pharmacol, 63, 538-546. Structural Analysis of the Activation of Ribavirin Analogs by NDP Kinase: Comparison with Other Ribavirin Targets. DOI:10.1124/mol.63.3.538. PMID:12606760.
  4. Hutter MC et al. (2002), Chembiochem, 3, 643-651. The Mechanism of Phosphorylation of Natural Nucleosides and Anti-HIV Analogues by Nucleoside Diphosphate Kinase Is Independent of Their Sugar Substituents. DOI:10.1002/1439-7633(20020703)3:7<643::aid-cbic643>3.0.co;2-l. PMID:12324998.
  5. Gonin P et al. (1999), Biochemistry, 38, 7265-7272. Catalytic Mechanism of Nucleoside Diphosphate Kinase Investigated Using Nucleotide Analogues, Viscosity Effects, and X-ray Crystallography†,‡. DOI:10.1021/bi982990v. PMID:10353838.
  6. Admiraal SJ et al. (1999), Biochemistry, 38, 4701-4711. Nucleophilic Activation by Positioning in Phosphoryl Transfer Catalyzed by Nucleoside Diphosphate Kinase†,‡. DOI:10.1021/bi9827565. PMID:10200157.
  7. Moréra S et al. (1995), Biochemistry, 34, 11062-11070. Mechanism of phosphate transfer by nucleoside diphosphate kinase: X-ray structures of the phosphohistidine intermediate of the enzymes from Drosophila and Dictyostelium. DOI:10.2210/pdb1nsp/pdb. PMID:7669763.

Step 1. The gamma phosphate of ATP deprotonates His122. His122 then attacks the gamma phosphate of ATP in a nucleophilic substitution, releasing the ADP product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His122B hydrogen bond donor
Lys16B hydrogen bond donor, electrostatic stabiliser
Tyr56B hydrogen bond donor, electrostatic stabiliser
Asn119B hydrogen bond donor, steric role, hydrogen bond acceptor, electrostatic stabiliser
Glu133B hydrogen bond acceptor, increase nucleophilicity, steric role
His122B nucleophile, proton donor

Chemical Components

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

Step 2. The nucleoside diphosphate substrate attacks the phosphate of the phosphorylated His122 in a nucleophilic substitution. The released His122 then deprotonates the triphosphate giving the nucleoside triphosphate product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys16B hydrogen bond donor, electrostatic stabiliser
Tyr56B hydrogen bond donor, electrostatic stabiliser
Asn119B hydrogen bond donor, steric role, hydrogen bond acceptor, electrostatic stabiliser
Glu133B hydrogen bond acceptor, steric role
His122B proton acceptor, nucleofuge

Chemical Components

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

Step 1. The gamma phosphate of ATP deprotonates His122. His122 then attacks the gamma phosphate of ATP in a nucleophilic substitution, releasing the ADP product.

Step 2. The nucleoside diphosphate substrate attacks the phosphate of the phosphorylated His122 in a nucleophilic substitution. The released His122 then deprotonates the triphosphate giving the nucleoside triphosphate product.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Alex Gutteridge, Craig Porter, Jonathan T. W. Ng, Charity Hornby