Enzyme - 5'-nucleotidase

Alternative Name(s)

There are no alternative names for this Enzyme

Catalytic Activity

ribonucleoside 5'-phosphate + H2O = a ribonucleoside + phosphate


There are no Cofactors for this Enzyme

Reaction Mechanisms

    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.

    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.


    The products of the reaction.

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

Protein name Organism
Cytosolic purine 5'-nucleotidase Bovine
Probable 5'-nucleotidase Treponema pallidum (strain Nichols)
5'-nucleotidase SurE1 Pyrobaculum aerophilum (strain ATCC 51768 / IM2 / DSM 7523 / JCM 9630 / NBRC 100827)
Pyrimidine 5'-nucleotidase YjjG Escherichia coli (strain K12)
5'-nucleotidase Human