Cofactors

There are no Cofactors for this Enzyme

Reaction Mechanisms

5'-nucleotidase (bacterial) By Reference

5'-nucleotidase is a zinc-containing enzyme involved in ATP hydrolysis and is bound to the membrane as an extracellular nucleotidase, or ectonucleotidase. Bacterial 5'-nucleotidases show a significant sequence homology to animal counterparts suggesting a common evolutionary origin. It is also related to 2',3'-cyclic phosphodiesterases and apyrases, all being part of the superfamily of metallophosphoesterases which also includes the Ser/Thr protein phosphatases and the purple acid phosphatases. The enzyme also has a UDP-sugar hydrolase activity which catalyses the periplasmic degradation of external UDP-glucose to uridine, glucose-1-phosphate and phosphate.




• Summary
• Step 1
• Step 2
• Step 3
• Products

5'-nucleotidase catalyses the hydrolytic cleavage of 5'-mononucleotides to nucleoside and phosphate by nucleophilic attack of a water molecule on the phosphorous. A water ligand is coordinated to the di-zinc centre. The phosphate group of the substrate binds with one oxygen atom to a metal ion. The metal-bound water attacks the phosphorous nucleophilically, with the proton transferred to His 117. The pentahedral transition state is stabilised by both metal ions, Asn 116, Arg 375, Arg 379, Arg 410 and His 117. After expulsion of the leaving group the tetrahedral phosphate ion remains bound to the dimetal centre.

Catalytic Residues

AA	Uniprot	Uniprot Resid	PDB	PDB Resid
Arg	P07024	379	1ush	379
Asp	P07024	41	1ush	41
Asp	P07024	84	1ush	84
His	P07024	217	1ush	217
His	P07024	252	1ush	252
Gln	P07024	254	1ush	254
His	P07024	43	1ush	43
Asn	P07024	116	1ush	116
His	P07024	117	1ush	117
Asp	P07024	120	1ush	120
Arg	P07024	375	1ush	375
Arg	P07024	410	1ush	410

Step Components

heterolysis, coordination to a metal ion, overall reactant used, bimolecular nucleophilic addition, decoordination from a metal ion, proton transfer, overall product formed



Step 1.

His117/Asp120 dyad and the two zinc ions activate a water molecule to perform a nucleophiliic attack on the phosphate group. This forms a penta-covalent intermediate stabilized by the arginine residues. The bridging water is thought to be the nucleophile as it has the lowest pKa out of the potential water molecules.



Step 2.

Since no acidic residues are positioned to protonate the leaving group this role is thought to be performed by a solvent water molecule. This leaves a tetrahedral phosphate bound to the zinc ions.



Step 3.

Phosphate leaves the active site and the active site transitions to its resting state with each zinc ion being 5 coordinate and no terminal water molecules coordianted.



Products.

The products of the reaction.

View All 2 Reaction Mechanisms in M-CSA

Reaction Parameters

• Kinetic Parameters

  Organism	KM Value [mM]	Substrate	Comment
  Escherichia coli	5.4	ATP	N-terminal protein domain, pH 7.0, 22°C
  Bacillus subtilis	37	4-nitrophenyl phosphate	at pH 8.9 and 25°C
  Homo sapiens	48800	5'-AMP	37°C, pH not specified in the publication
  Mus musculus	0.01	5'-UMP	mutant W11A, pH 7.5, 37°C
  Legionella pneumophila	1.7	5'-GMP	sigmoidal kinetic profile, pH 8.0, temperature not specified in the publication

• Temperature

  Organism	Temperature Range	Comment
  Streptococcus pyogenes	37 - 47

• pH

  Organism	pH Range	Comment
  Streptococcus pyogenes	5 - 6.5	the optimal enzymatic activity is between pH 5.0 and pH 6.5. The activity decreases at higher pH but is still at about 25% at pH 9.0
  Arachis hypogaea	5 - 6
  Rhipicephalus microplus	5.8 - 8.5
  Salinivibrio costicola	7 - 9	less than 50% of maximal activity above and below

Associated Proteins

Citations

• Novel microRNAs modulating ecto-5'-nucleotidase expression.
• Anti-inflammatory potency of novel ecto-5'-nucleotidase/CD73 inhibitors in astrocyte culture model of neuroinflammation.
• Expression of functionally distinct ecto-5'-nucleotidase/CD73 glycovariants in reactive astrocytes in experimental autoimmune encephalomyelitis and neuroinflammatory conditions in vitro.
• Notch1 promotes resistance to cisplatin by up-regulating Ecto-5'-nucleotidase (CD73) in triple-negative breast cancer cells.
• A rare mutation (p.F149del) of the NT5C3A gene is associated with pyrimidine 5'-nucleotidase deficiency.
• Molecular Dynamics Simulations of the Human Ecto-5'-Nucleotidase (h-ecto-5'-NT, CD73): Insights into Protein Flexibility and Binding Site Dynamics.
• Probiotic-Derived Ecto-5'-Nucleotidase Produces Anti-Inflammatory Adenosine Metabolites in Treg-Deficient Scurfy Mice.
• Anti-Cytosolic 5'-Nucleotidase 1A in the Diagnosis of Patients with Suspected Idiopathic Inflammatory Myopathies: An Italian Real-Life, Single-Centre Retrospective Study.
• Mesenchymal Stromal Cell Exosomes Mediate M2-like Macrophage Polarization through CD73/Ecto-5'-Nucleotidase Activity.
• Probiotic-derived ecto-5'-nucleotidase produces anti-inflammatory adenosine metabolites in Treg-deficient scurfy mice
• Myositis-related autoantibody profile and clinical characteristics stratified by anti-cytosolic 5'-nucleotidase 1A status in connective tissue diseases.